Methods for spatial analysis using targeted RNA depletion

ABSTRACT

Provided herein are methods for spatial analysis using targeted RNA depletion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/546,625, filed on Dec. 9, 2021, which is a continuation ofInternational Application PCT/US2021/028397, with an internationalfiling date of Apr. 21, 2021, which claims priority to U.S. ProvisionalPatent Application No. 63/014,054, filed Apr. 22, 2020, which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an XML file named “47706-0200002_SL_ST26.xml”. The XMLfile, created on Dec. 21, 2022, is 178,865 bytes in size. The materialin the XML file is hereby incorporated by reference in its entirety.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphologyand/or function due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, and signaling and cross-talk with other cellsin the tissue.

Spatial heterogeneity has been previously studied using techniques thatonly provide data for a small handful of analytes in the context of anintact tissue or a portion of a tissue, or provide a lot of analyte datafor single cells, but fail to provide information regarding the positionof the single cell in a parent biological sample (e.g., tissue sample).

Undesirable RNA (e.g, ribosomal RNA) constitutes a considerableproportion of the total nucleic acid pool from the biological sample,which can compete with hybridization of target analytes of interest. Incertain settings, undesirable RNA is capable of hybridizing torandomers, poly-adenylated sequences, and even gene specific capturesequences, thus creating increased background signal that interfereswith target analyte binding.

One option for decreasing background signal of undesirable RNA moleculesis to add depletion probes during the reverse transcription step of thespatial array gene expression protocol. Depletion probes can be designedto tile various types of undesirable RNA molecules (e.g., both nuclearand mitochondrial RNA molecules). In this setting, by tiling rRNAmolecules, the molecules are largely inhibited from interacting with thespatial capture array. However, one drawback could be that it ispossible that a considerable fraction of rRNA are already interactingwith the array when the depletion probes are added, thereby limitedtheir utility. Thus, there is a need to remove such undesirable RNA.

RNA-templated ligation (RTL) is the process by which multipleoligonucleotides hybridize to an analyte at nearby or adjacent sequencesfollowed by ligation of the oligonucleotides to create a ligationproduct. After hybridization and ligation, a DNA-RNA hybrid complex thatincludes the analyte of interest (e.g., RNA) and the ligated probe (madeof DNA or DNA/RNA combination) is created. RTL utilizes a ribonuclease(e.g., RNAse H) to digest the DNA-RNA hybrid complex, freeing theligated probe for downstream applications such as spatial array probehybridization and sequencing. Here, Applicants have identified thatundesirable RNA probes can also be added and hybridized to undesirableRNAs. Further, because the undesirable RNA probes/undesirable RNAcomplex creates a DNA-RNA hybrid complex, the same endonuclease step candigest the analyte also digests the undesirable RNA. Because thereremains a need to remove such undesirable RNA, this approach simplifiesthe necessity for multiple enzymatic steps that could affect nucleicacid integrity and function.

SUMMARY

The present invention relates to methods of depleting undesirable RNAfrom nucleic acid samples. The invention is useful for preparing cDNAfrom the RNA-depleted nucleic acid samples, for example, from fixedparaffin embedded (FFPE) tissue samples.

In one aspect, provided herein is a method for identifying a location ofan analyte in a biological sample, the method comprising: (a) contactinga biological sample with a first probe oligonucleotide, a second probeoligonucleotide, and a plurality of undesirable RNA depletion probes,wherein the first probe oligonucleotide and the second probeoligonucleotide are substantially complementary to adjacent sequences ofthe analyte, wherein the second probe oligonucleotide comprises acapture probe binding domain that is capable of binding to a capturedomain of a capture probe, and wherein an undesirable RNA depletionprobe of the plurality of undesirable RNA depletion probes issubstantially complementary to a sequence of an undesirable RNA moleculein the biological sample; (b) hybridizing the first probeoligonucleotide and the second probe oligonucleotide to the analyte; (c)hybridizing the undesirable RNA depletion probe to the undesirable RNAmolecule; (d) ligating the first probe oligonucleotide and the secondprobe oligonucleotide, thereby creating a ligated probe that issubstantially complementary to the analyte; (e) removing the pluralityof undesirable RNA depletion probe-undesirable RNA complexes andreleasing the ligated probe from the analyte; (f) hybridizing thecapture probe binding domain of the ligated probe to a capture domain ofa capture probe that is affixed to the substrate; and (g) determining(i) all or a part of the sequence of the ligated probe specificallybound to the capture domain, or a complement thereof, and (ii) all or apart of the sequence of the spatial barcode, or a complement thereof,and using the determined sequence of (i) and (ii) to identify thelocation of the analyte in the biological sample.

In some embodiments, the first probe oligonucleotide comprises at leasttwo ribonucleic acid bases at the 3′ end.

In some embodiments, the first probe oligonucleotide further comprises afunctional sequence. In some embodiments, the functional sequence is aprimer sequence.

In some embodiments, the second probe oligonucleotide comprises aphosphorylated nucleotide at the 5′ end.

In some embodiments, the method further comprises providing a captureprobe binding domain blocking moiety that interacts with the captureprobe binding domain.

In some embodiments, the method further comprises releasing the captureprobe binding domain blocking moiety from the capture probe bindingdomain prior to step (f).

In some embodiments, the capture probe binding domain comprises apoly-adenylated (poly(A)) sequence or a complement thereof.

In some embodiments, the capture probe binding domain blocking moietycomprises a poly-uridine sequence, a poly-thymidine sequence, or both.

In some embodiments, releasing the poly-uridine sequence from thepoly(A) sequence comprises denaturing the ligated probe or contactingthe ligated probe with an endonuclease or exonuclease.

In some embodiments, the capture probe binding domain comprises asequence that is complementary to all or a portion of the capture domainof the capture probe. In some embodiments, the capture probe bindingdomain comprises a degenerate sequence.

In some embodiments, the ligation step comprises ligating the first andsecond probe oligonucleotides using enzymatic ligation or chemicalligation. In some embodiments, the enzymatic ligation utilizes a ligase.

In some embodiments, the ligase is one or more of a T4 RNA ligase(Rnl2), a splintR ligase, a single stranded DNA ligase, or a T4 DNAligase. In some embodiments, the ligase is a T4 RNA ligase 2 (Rnl2)ligase.

In some embodiments, the first probe oligonucleotide and the secondprobe oligonucleotide are DNA probes. In some embodiments, theundesirable RNA depletion probe is a DNA probe.

In some embodiments, the steps (b) and (c) each creates a RNA: DNAhybrid.

In some embodiments, step (e) comprises contacting the undesirable RNAdepletion probe with a ribonuclease.

In some embodiments, the ribonuclease is RNase H. In some embodiments,the RNase H is RNase H1. In some embodiments, the RNase H is RNase H2.In some embodiments, the RNase H is a thermostable RNase.

In some embodiments, the method further comprises amplifying the ligatedprobe prior to step (f). In some embodiments, steps (b) and (c) areperformed at substantially the same time.

In one aspect, provided herein is a method for identifying a location ofan analyte in a biological sample, the method comprising: (a) contactingthe biological sample with a substrate comprising a plurality ofattached capture probes, wherein a capture probe of the pluralitycomprises (i) the spatial barcode and (ii) a capture domain that bindsspecifically to a sequence present in the analyte; and a plurality ofundesirable RNA depletion probes, wherein an undesirable RNA depletionprobe in the plurality of undesirable RNA depletion probes issubstantially complementary to a sequence of an undesirable RNA moleculein the biological sample; (b) hybridizing the undesirable RNA depletionprobe to the undesirable RNA; (c) removing the plurality of undesirableRNA depletion probe-undesirable RNA complexes; (d) hybridizing theanalyte to a capture domain of a capture probe that is affixed to thesubstrate; (e) extending a 3′ end of the capture probe using the analytethat is specifically bound to the capture domain as a template togenerate an extended capture probe; and (f) amplifying the extendedcapture probe to produce a nucleic acid.

In some embodiments, provided herein is the method for identifying alocation of an analyte in a biological sample, further comprisingdetermining (i) all or a part of the sequence of the spatial barcode orthe complement thereof, and (ii) all or a portion of the sequence of theanalyte from the biological sample, and using the determined sequencesof (i) and (ii) to identify the location of the analyte in thebiological sample.

In one aspect, provided herein is a method for identifying a location ofan analyte in a biological sample, the method comprising: (a) contactingthe biological sample with a plurality of undesirable RNA depletionprobes, wherein an undesirable RNA depletion probe in the plurality ofundesirable RNA depletion probes is substantially complementary to asequence of an undesirable RNA molecule in the biological sample; (b)hybridizing the undesirable RNA depletion probe to the undesirable RNA;(c) removing the plurality of undesirable RNA depletionprobe-undesirable RNA complexes; (d) contacting a plurality of nucleicacids with a plurality of target oligonucleotide probes, wherein anucleic acid of the plurality of nucleic acids comprises (i) a spatialbarcode or a complement thereof, and (ii) a portion of a sequence of ananalyte from a biological sample, or a complement thereof; and a targetoligonucleotide probe of the plurality of target oligonucleotide probescomprises: a domain that binds specifically to (i) all or a portion ofthe spatial barcode or a complement thereof, and/or (ii) all or aportion of the sequence of the analyte from the biological sample, or acomplement thereof, and a molecular tag; (e) enriching a complex of thetarget oligonucleotide probe specifically bound to the nucleic acidusing a substrate comprising an agent that binds specifically to themolecular tag; and (f) determining (i) all or a portion of the sequenceof the spatial barcode or the complement thereof, and (ii) all or aportion of the sequence of the analyte from the biological sample, andusing the determined sequences of (i) and (ii) to identify the locationof the analyte in the biological sample.

In some embodiments, the method further comprises generating theplurality of nucleic acids comprises: (a) contacting the biologicalsample with a substrate comprising a plurality of attached captureprobes, wherein a capture probe of the plurality comprises (i) thespatial barcode and (ii) a capture domain that binds specifically to asequence present in the analyte; (b) extending a 3′ end of the captureprobe using the analyte that is specifically bound to the capture domainas a template to generate an extended capture probe; and (c) amplifyingthe extended capture probe to produce the nucleic acid.

In some embodiments, the domain of the target oligonucleotide probecomprises a total of about 40 nucleotides to about 160 nucleotides.

In some embodiments, the molecular tag comprises a moiety. In someembodiments, the moiety is streptavidin, avidin, biotin, or afluorophore.

In some embodiments, the molecular tag comprises a small molecule, anucleic acid, or a carbohydrate.

In some embodiments, the molecular tag is positioned 5′ or 3′ to thedomain in the target oligonucleotide probe.

In some embodiments, the agent that binds specifically to the moleculartag comprises a protein. In some embodiments, the protein is anantibody.

In some embodiments, the agent that binds specifically to the moleculartag comprises a nucleic acid. In some embodiments, the nucleic acid isDNA.

In some embodiments, the agent that binds specifically to the moleculartag comprises a small molecule.

In some embodiments, the analyte from the biological sample isassociated with a disease or condition. In some embodiments, the analytefrom the biological sample comprises a mutation. In some embodiments,the analyte from the biological sample comprises a single nucleotidepolymorphism (SNP). In some embodiments, the analyte from the biologicalsample comprises a trinucleotide repeat.

In some embodiments, the biological sample is a tissue sample.

In some embodiments, the tissue sample is a formalin-fixed,paraffin-embedded (FFPE) tissue sample, a fresh or a frozen tissuesample. In some embodiments, the tissue sample is the FFPE tissuesample, and the tissue sample is decrosslinked.

In some embodiments, the biological sample was previously stained. Insome embodiments, the biological sample was previously stained usinghematoxylin and eosin (H&E). In some embodiments, the biological samplewas previously stained using immunofluorescence or immunohistochemistry.

In some embodiments, the method further comprises contacting thebiological sample with a permeabilization agent.

In some embodiments, the biological sample is a permeabilized biologicalsample that has been permeabilized with a permeabilization agent.

In some embodiments, the permeabilization agent is selected from anorganic solvent, a detergent, and an enzyme, or a combination thereof.In some embodiments, the permeabilization agent is an endopeptidase orprotease. In some embodiments, the endopeptidase is pepsin or proteinaseK.

In some embodiments, the determining step comprises amplifying all orpart of the ligated probe specifically bound to the capture domain.

In some embodiments, the amplifying is isothermal. In some embodiments,the amplifying is not isothermal.

In some embodiments, an amplifying product comprises (i) all or part ofsequence of the ligated probe specifically bound to the capture domain,or a complement thereof, and (ii) all or a part of the sequence of thespatial barcode, or a complement thereof.

In some embodiments, the determining step comprises sequencing.

In some embodiments, the analyte is RNA. In some embodiments, the RNA isan mRNA.

In one aspect, provided herein is a method for enriching a targetnucleic acid in a spatial array comprising (a) adding a plurality ofundesirable RNA depletion probes to the spatial array, wherein anundesirable RNA depletion probe of the plurality of undesirable RNAdepletion probes is substantially complementary to a sequence of anundesirable RNA molecule in the spatial array; (b) hybridizing anundesirable RNA depletion probe to the undesirable RNA; (c) removing theplurality of undesirable RNA depletion probe-undesirable RNA complexes;and (d) amplying remaining nucleic acids to enrich the target nucleiacid.

In one aspect, provided herein is a method for depleting undesirable RNAmolecules in a spatial array, comprising (a) adding a plurality ofundesirable RNA depletion probes to the spatial array, wherein anundesirable RNA depletion probe of the plurality of undesirable RNAdepletion probes is substantially complementary to a sequence of anundesirable RNA molecule in the spatial array; (b) hybridizing anundesirable RNA depletion probe to the undesirable RNA; and (c) removingthe plurality of undesirable RNA depletion probe-undesirable RNAcomplexes to deplete the undesirable RNA molecules.

In some embodiments, the undesirable RNA depletion probe is a DNA probe.

In some embodiments, the hybridizing step comprises hybridizing the DNAprobe with the undesirable RNA molecule that creates a RNA: DNA hybrid.

In some embodiments, the removing step comprises contacting theundesirable RNA depletion probe with a ribonuclease.

In some embodiments, the ribonuclease is RNase H. In some embodiments,the RNase H is RNase H1. In some embodiments, the RNase H is RNase H2.In some embodiments, the RNase H is a thermostable RNase.

In some embodiments, the undesirable RNA depletion probe issubstantially complementary to all or a portion of the sequence of theundesirable RNA molecule in the biological sample.

In some embodiments, at least one undesirable RNA depletion probespecifically hybridizes to substantially one or more portions of thesequence of the undesirable RNA molecule.

In some embodiments, at least one undesirable RNA depletion probespecifically hybridizes to substantially the entire full length sequenceof the undesirable RNA molecule.

In some embodiments, the undesirable RNA molecule is a transfer RNA(tRNA), a ribosomal RNA (rRNA), a messenger RNA (mRNA), or anycombinations thereof.

In some embodiments, the undesirable RNA molecule is a mitochondrialRNA, nuclear RNA, or cytoplasmic RNA.

In some embodiments, the undesirable RNA depletion probe furthercomprises a capture moiety. In some embodiments, the removing stepcomprises using a capture moiety-binding agent that binds specificallyto the capture moiety.

In some embodiments, the capture moiety is streptavidin, avidin, biotin,or a fluorophore. In some embodiments, the capture moiety is a biotin.

In some embodiments, the capture moiety comprises a small molecule, anucleic acid, or a carbohydrate.

In some embodiments, the capture moiety is positioned 5′ or 3′ to thedomain in the undesirable RNA depletion probe.

In some embodiments, a capture moiety-binding agent that bindsspecifically to the capture moiety comprises a protein.

In some embodiments, the protein is an antibody. In some embodiments,the protein is streptavidin.

In some embodiments, the capture moiety-binding agent that bindsspecifically to the capture moiety comprises a nucleic acid. In someembodiments, the nucleic acid is DNA.

In some embodiments, the capture moiety-binding agent that bindsspecifically to the capture moiety comprises a small molecule.

In some embodiments, the capture moiety-binding agent that bindsspecifically to the capture moiety is attached to a substrate.

In some embodiments, the substrate is a bead. In some embodiments, thebead is a magnetic bead. In some embodiments, the capture moiety is abiotin and the capture moiety-binding agent is streptavidin. In someembodiments, the streptavidin is attached to a magnetic bead that allowsthe undesirable RNA depletion probe-undesirable RNA complexes to beremoved magnetically from the biological sample.

In some embodiments, the capture probes are capable of hybridizing tothe ligated probe as described herein.

In some embodiments, the capture probes further comprises a functionalsequence. In some embodiments, the functional sequence is primersequence or a complement thereof. In some embodiments, the capture probefurther comprises a unique molecular sequence or a complement thereof.In some embodiments, the capture probe further comprises an additionalprimer binding sequence or a complement thereof.

In one aspect, provided herein is a kit comprising (a) an arraycomprising a plurality of capture probes; (b) a plurality of probeoligonucleotides comprising a first probe oligonucleotide and a secondoligonucleotide, wherein the first probe oligonucleotide and the secondprobe oligonucleotide are substantially complementary to adjacentsequences of an analyte, wherein the second probe oligonucleotidecomprises a capture probe binding domain that is capable of binding to acapture domain of the capture probe; (c) a plurality of enzymescomprising a ribonuclease and a ligase; and (d) an instruction for usingthe kit.

In some embodiments, the ribonuclease is RNase H.

In some embodiments, the ligase is one or more of a T4 RNA ligase(Rnl2), a splintR ligase, a single stranded DNA ligase, or a T4 DNAligase. In some embodiments, the ligase is a T4 RNA ligase 2 (Rnl2)ligase.

All publications, patents, patent applications, and informationavailable on the internet and mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication, patent, patent application, or item of information wasspecifically and individually indicated to be incorporated by reference.To the extent publications, patents, patent applications, and items ofinformation incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 2 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to target analytes within the sample.

FIG. 3 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature.

FIG. 4 is a schematic diagram of an exemplary analyte capture agent.

FIG. 5 is a schematic diagram depicting an exemplary interaction betweena feature-immobilized capture probe 524 and an analyte capture agent526.

FIGS. 6A, 6B, and 6C are schematics illustrating how streptavidin celltags can be utilized in an array-based system to producespatially-barcoded cells or cellular contents.

FIG. 7 shows a schematic workflow illustrating exemplary, non-limiting,non-exhaustive steps for in situ ribosomal RNA depletion.

FIG. 8A shows a schematic illustrating an exemplary workflow forribosomal depletion (RD).

FIG. 8B shows an H&E staining image and an 18S rRNA staining image. Noribosomal depletion was performed.

FIG. 8C shows an H&E staining image and an 18S rRNA staining image.Ribosomal depletion was performed using RD probes designed to block boththe cytoplasmic RNA (18S, 28S, 5S and 5.8S) and mitochondrial RNA (16Sand 12S).

FIG. 8D shows an H&E staining image and an mRNA staining image usingpolyA probes. No ribosomal depletion was performed.

FIG. 8E shows an H&E staining image and an mRNA staining image usingpolyA probes. Ribosomal depletion was performed using RD probes designedto block both the cytoplasmic RNA (18S, 28S, 5S and 5.8S) andmitochondrial RNA (16S and 12S).

FIG. 9A shows the gene-gene scatter plot between normal and ribosomaldepleted mouse olfactory bulb (MOB) tissues. Ribosomal depletion wasperformed using RD probes designed to block both the cytoplasmic RNA(18S, 28S, 5S and 5.8S) and mitochondrial RNA (16S and 12S).

FIG. 9B shows the gene-gene scatter plot between normal and ribosomaldepleted childhood brain cancer (PNET) tissues. Ribosomal depletion wasperformed using RD probes designed to block both the cytoplasmic RNA(18S, 28S, 5S and 5.8S) and mitochondrial RNA (16S and 12S).

FIG. 9C shows the gene-gene scatter plot between normal and ribosomaldepleted adipose (fat) tissues. Ribosomal depletion was performed usingRD probes designed to block both the cytoplasmic RNA (18S, 28S, 5S and5.8S) and mitochondrial RNA (16S and 12S).

FIG. 10 shows tissue plots illustrating the gene expression level ofMT-RNR1 or MT-RNR2 of normal or ribosomal depleted tissues. Ribosomaldepletion was performed using RD probes designed to block both thecytoplasmic RNA (18S, 28S, 5S and 5.8S) and mitochondrial RNA (16S and12S).

FIG. 11 shows the UMIs per gene in normal or ribosomal depleted tissues.The tissues include adipose (fat), mouse olfactory bulb (MOB),MOB-181218, and childhood brain cancer (PNET) tissues.

FIG. 12 shows the detection rate comparison between normal and ribosomaldepleted tissues. The tissues include adipose (fat), mouse olfactorybulb (MOB), MOB-181218, and childhood brain cancer (PNET) tissues.

FIG. 13A shows tissue plots by Seurat clustering for 7 clusters from anormal tissue.

FIG. 13B shows tissue plots by Seurat clustering for 7 clusters from aribosomal depleted tissue.

FIG. 14A shows a tSNE plot of Seurat clustering corresponding to thetissue plots in FIG. 13A.

FIG. 14B shows a tSNE plot of Seurat clustering corresponding to thetissue plots in FIG. 13B.

FIG. 15 shows tissue plots of 7 clusters from a normal tissue (sameclusters from FIG. 14A).

FIG. 16 shows tissue plots of 7 clusters from a ribosomal depletedtissue (same clusters from FIG. 14B).

FIG. 17A shows tissue plots of 8 clusters from a normal tissuecorresponding to the Seurat clusters in the tSNE plot in FIG. 17B. Thetwo arrows indicate clusters 1 and 5 (also indicated by numerals).

FIG. 17B shows a tSNE plot of Seurat clustering corresponding to theindicated tissue plots in FIG. 17A. The two arrows indicate clusters 1and 5 (also indicated by numerals).

FIG. 18A shows tissue plots of 8 clusters from a ribosomal depletedtissue corresponding to the Seurat clusters in the tSNE plot in FIG.18B. The two arrows indicate clusters 3 and 4, (also indicated bynumerals).

FIG. 18B shows a tSNE plot of Seurat clustering corresponding to theindicated tissue plots in FIG. 18A. The two arrows indicate clusters 3and 4 (also indicated by numerals).

FIGS. 19A-19D show H&E staining images and gene expression heat maps forcontrol samples (samples 1 and 2) and ribosomal depletion samples(samples 3 and 4). FIGS. 19B-19D show gene expression heat maps forsamples 1˜4 for Penk, Doc2g, and Kctd12, respectively.

FIGS. 20A-20D show gene expression heat maps for control samples (sample1 and 2) and ribosomal depletion samples (samples 3 and 4). FIGS. 20Aand 20B show gene expression heat maps for house keeping genes: Actb andGapdh, respectively. FIGS. 20C and 20D show gene expression heat mapsfor two targets of the ribosomal depletion probes: mt-Rnr1 and mt-Rnr2,respectively.

DETAILED DESCRIPTION

I. Introduction

Disclosed herein are methods and compositions predicated on usingtargeted RNA depletion to remove one or more species of undesirable RNAmolecules (e.g., ribosomal RNA and/or mitochondrial RNA) to reduce thepool and concentration of undesirable RNA molecules in a sample whichcould interfere with desired target detection (e.g., detection of mRNA).To achieve depletion, one or more probes are designed that hybridize toone or more undesirable RNA molecules. For example, in one embodiment,probes can be administered to a biological sample that selectivelyhybridize to ribosomal RNA (rRNA), thereby reducing the pool andconcentration of rRNA in the sample. Here, this type of RNA depletion iscombined with spatial analysis techniques in order to determineabundance and/or location of one or more analytes in a biologicalsample. The ability to reduce interference with detection of desiredtargets by removing undesirable RNA increases efficiency and sensitivity

+y of the spatial analysis techniques. For example, subsequent orconcurrent application of capture probes to the sample can result inimproved capture of other types of RNA (e.g., mRNA or products ofRNA-templated ligation) due to a reduction in undesirable RNA (e.g.,down-selected RNA) present in the sample.

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination. Further non-limitingaspects of spatial analysis methodologies and compositions are describedherein.

Some general terminology that may be used in this disclosure can befound in Section (I)(b) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Typically, a “barcode” is a label, oridentifier, that conveys or is capable of conveying information (e.g.,information about an analyte in a sample, a bead, and/or a captureprobe). A barcode can be part of an analyte, or independent of ananalyte. A barcode can be attached to an analyte. A particular barcodecan be unique relative to other barcodes. For the purpose of thisdisclosure, an “analyte” can include any biological substance,structure, moiety, or component to be analyzed. The term “target” cansimilarly refer to an analyte of interest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in Section (I)(c)of WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. In some embodiments, an analyte can be detectedindirectly, such as through detection of an intermediate agent, forexample, a connected probe (e.g., a ligation product) or an analytecapture agent (e.g., an oligonucleotide-conjugated antibody), such asthose described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in Section (I)(d) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in Section(I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some embodiments,a capture probe can include a cleavage domain and/or a functional domain(e.g., a primer-binding site, such as for next-generation sequencing(NGS)).

FIG. 1 is a schematic diagram showing an exemplary capture probe, asdescribed herein. As shown, the capture probe 102 is optionally coupledto a feature 101 by a cleavage domain 103, such as a disulfide linker.The capture probe can include a functional sequence 104 that is usefulfor subsequent processing. The functional sequence 104 can include allor a part of sequencer specific flow cell attachment sequence (e.g., aP5 or P7 sequence), all or a part of a sequencing primer sequence,(e.g., a R1 primer binding site, a R2 primer binding site), orcombinations thereof. The capture probe can also include a spatialbarcode 105. The capture probe can also include a unique molecularidentifier (UMI) sequence 106. While FIG. 1 shows the spatial barcode105 as being located upstream (5′) of UMI sequence 106, it is to beunderstood that capture probes wherein UMI sequence 106 is locatedupstream (5′) of the spatial barcode 105 is also suitable for use in anyof the methods described herein. The capture probe can also include acapture domain 107 to facilitate capture of a target analyte. Thecapture domain can have a sequence complementary to a sequence of anucleic acid analyte. The capture domain can have a sequencecomplementary to a connected probe described herein. The capture domaincan have a sequence complementary to a capture handle sequence presentin an analyte capture agent. The capture domain can have a sequencecomplementary to a splint oligonucleotide. Such splint oligonucleotide,in addition to having a sequence complementary to a capture domain of acapture probe, can have a sequence of a nucleic acid analyte, a sequencecomplementary to a portion of a connected probe described herein, and/ora capture handle sequence described herein.

The functional sequences can generally be selected for compatibilitywith any of a variety of different sequencing systems, e.g., Ion TorrentProton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore,etc., and the requirements thereof. In some embodiments, functionalsequences can be selected for compatibility with non-commercializedsequencing systems. Examples of such sequencing systems and techniques,for which suitable functional sequences can be used, include (but arenot limited to) Ion Torrent Proton or PGM sequencing, Illuminasequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.Further, in some embodiments, functional sequences can be selected forcompatibility with other sequencing systems, includingnon-commercialized sequencing systems.

In some embodiments, the spatial barcode 105 and functional sequences104 are common to all of the probes attached to a given feature. In someembodiments, the UMI sequence 106 of a capture probe attached to a givenfeature is different from the UMI sequence of a different capture probeattached to the given feature.

FIG. 2 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to analytes within the sample. The capture probe 201 contains acleavage domain 202, a cell penetrating peptide 203, a reporter molecule204, and a disulfide bond (—S—S—). 205 represents all other parts of acapture probe, for example a spatial barcode and a capture domain.

FIG. 3 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature. In FIG. 3 , the feature 301 can be coupledto spatially-barcoded capture probes, wherein the spatially-barcodedprobes of a particular feature can possess the same spatial barcode, buthave different capture domains designed to associate the spatial barcodeof the feature with more than one target analyte. For example, a featuremay be coupled to four different types of spatially-barcoded captureprobes, each type of spatially-barcoded capture probe possessing thespatial barcode 302. One type of capture probe associated with thefeature includes the spatial barcode 302 in combination with a poly(T)capture domain 303, designed to capture mRNA target analytes. A secondtype of capture probe associated with the feature includes the spatialbarcode 302 in combination with a random N-mer capture domain 304 forgDNA analysis. A third type of capture probe associated with the featureincludes the spatial barcode 302 in combination with a capture domaincomplementary to a capture handle sequence of an analyte capture agentof interest 305. A fourth type of capture probe associated with thefeature includes the spatial barcode 302 in combination with a capturedomain that can specifically bind a nucleic acid molecule 306 that canfunction in a CRISPR assay (e.g., CRISPR/Cas9). While only fourdifferent capture probe-barcoded constructs are shown in FIG. 3 ,capture-probe barcoded constructs can be tailored for analyses of anygiven analyte associated with a nucleic acid and capable of binding withsuch a construct. For example, the schemes shown in FIG. 3 can also beused for concurrent analysis of other analytes disclosed herein,including, but not limited to: (a) mRNA, a lineage tracing construct,cell surface or intracellular proteins and metabolites, and gDNA; (b)mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq)cell surface or intracellular proteins and metabolites, and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (c)mRNA, cell surface or intracellular proteins and/or metabolites, abarcoded labelling agent (e.g., the MHC multimers described herein), anda V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). Insome embodiments, a perturbation agent can be a small molecule, anantibody, a drug, an aptamer, a miRNA, a physical environmental (e.g.,temperature change), or any other known perturbation agents. See, e.g.,Section (II)(b) (e.g., subsections (i)-(vi)) of WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663. Generation ofcapture probes can be achieved by any appropriate method, includingthose described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in Section (IV) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) a capture handlesequence. As used herein, the term “analyte binding moiety barcode”refers to a barcode that is associated with or otherwise identifies theanalyte binding moiety. As used herein, the term “analyte capturesequence” or “capture handle sequence” refers to a region or moietyconfigured to hybridize to, bind to, couple to, or otherwise interactwith a capture domain of a capture probe. In some embodiments, a capturehandle sequence is complementary to a capture domain of a capture probe.In some cases, an analyte binding moiety barcode (or portion thereof)may be able to be removed (e.g., cleaved) from the analyte captureagent.

FIG. 4 is a schematic diagram of an exemplary analyte capture agent 402comprised of an analyte-binding moiety 404 and an analyte-binding moietybarcode domain 408. The exemplary analyte-binding moiety 404 is amolecule capable of binding to an analyte 406 and the analyte captureagent is capable of interacting with a spatially-barcoded capture probe.The analyte-binding moiety can bind to the analyte 406 with highaffinity and/or with high specificity. The analyte capture agent caninclude an analyte-binding moiety barcode domain 408, a nucleotidesequence (e.g., an oligonucleotide), which can hybridize to at least aportion or an entirety of a capture domain of a capture probe. Theanalyte-binding moiety barcode domain 408 can comprise an analytebinding moiety barcode and a capture handle sequence described herein.The analyte-binding moiety 404 can include a polypeptide and/or anaptamer. The analyte-binding moiety 404 can include an antibody orantibody fragment (e.g., an antigen-binding fragment).

FIG. 5 is a schematic diagram depicting an exemplary interaction betweena feature-immobilized capture probe 524 and an analyte capture agent526. The feature-immobilized capture probe 524 can include a spatialbarcode 508 as well as functional sequences 506 and UMI 510, asdescribed elsewhere herein. The capture probe can also include a capturedomain 512 that is capable of binding to an analyte capture agent 526.The analyte capture agent 526 can include a functional sequence 518,analyte binding moiety barcode 516, and a capture handle sequence 514that is capable of binding to the capture domain 512 of the captureprobe 524. The analyte capture agent can also include a linker 520 thatallows the capture agent barcode domain 516 to couple to the analytebinding moiety 522.

FIGS. 6A, 6B, and 6C are schematics illustrating how streptavidin celltags can be utilized in an array-based system to produce aspatially-barcoded cell or cellular contents. For example, as shown inFIG. 6A, peptide-bound major histocompatibility complex (MHC) can beindividually associated with biotin (β2m) and bound to a streptavidinmoiety such that the streptavidin moiety comprises multiple pMHCmoieties. Each of these moieties can bind to a TCR such that thestreptavidin binds to a target T-cell via multiple MHC/TCR bindinginteractions. Multiple interactions synergize and can substantiallyimprove binding affinity. Such improved affinity can improve labellingof T-cells and also reduce the likelihood that labels will dissociatefrom T-cell surfaces. As shown in FIG. 6B, a capture agent barcodedomain 601 can be modified with streptavidin 602 and contacted withmultiple molecules of biotinylated MHC 603 such that the biotinylatedMHC 603 molecules are coupled with the streptavidin conjugated captureagent barcode domain 601. The result is a barcoded MHC multimer complex605. As shown in FIG. 6B, the capture agent barcode domain sequence 601can identify the MHC as its associated label and also includes optionalfunctional sequences such as sequences for hybridization with otheroligonucleotides. As shown in FIG. 6C, one example oligonucleotide iscapture probe 606 that comprises a complementary sequence (e.g., rGrGrGcorresponding to C C C), a barcode sequence and other functionalsequences, such as, for example, a UMI, an adapter sequence (e.g.,comprising a sequencing primer sequence (e.g., R1 or a partial R1(“pR1”), R2), a flow cell attachment sequence (e.g., P5 or P7 or partialsequences thereof)), etc. In some cases, capture probe 606 may at firstbe associated with a feature (e.g., a gel bead) and released from thefeature. In other embodiments, capture probe 606 can hybridize with acapture agent barcode domain 601 of the MHC-oligonucleotide complex 605.The hybridized oligonucleotides (Spacer C C C and Spacer rGrGrG) canthen be extended in primer extension reactions such that constructscomprising sequences that correspond to each of the two spatial barcodesequences (the spatial barcode associated with the capture probe, andthe barcode associated with the MHC-oligonucleotide complex) aregenerated. In some cases, one or both of the corresponding sequences maybe a complement of the original sequence in capture probe 606 or captureagent barcode domain 601. In other embodiments, the capture probe andthe capture agent barcode domain are ligated together. The resultingconstructs can be optionally further processed (e.g., to add anyadditional sequences and/or for clean-up) and subjected to sequencing.As described elsewhere herein, a sequence derived from the capture probe606 spatial barcode sequence may be used to identify a feature and thesequence derived from spatial barcode sequence on the capture agentbarcode domain 601 may be used to identify the particular peptide MHCcomplex 604 bound on the surface of the cell (e.g., when usingMHC-peptide libraries for screening immune cells or immune cellpopulations).

Additional description of analyte capture agents can be found in Section(II)(b)(ix) of WO 2020/176788 and/or Section (II)(b)(viii) U.S. PatentApplication Publication No. 2020/0277663.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). Another method is to cleavespatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a connected probe (e.g., a ligation product)or an analyte capture agent), or a portion thereof), or derivativesthereof (see, e.g., Section (II)(b)(vii) of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663 regarding extendedcapture probes). In some cases, capture probes may be configured to forma connected probe (e.g., a ligation product) with a template (e.g., aDNA or RNA template, such as an analyte or an intermediate agent, orportion thereof), thereby creating ligations products that serve asproxies for a template.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended usingreverse transcription. In some embodiments, the capture probe isextended using one or more DNA polymerases. The extended capture probesinclude the sequence of the capture probe and the sequence of thespatial barcode of the capture probe.

In some embodiments, extended capture probes are amplified (e.g., inbulk solution or on the array) to yield quantities that are sufficientfor downstream analysis, e.g., via DNA sequencing. In some embodiments,extended capture probes (e.g., DNA molecules) act as templates for anamplification reaction (e.g., a polymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in Section (II)(a) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.Analysis of captured analytes (and/or intermediate agents or portionsthereof), for example, including sample removal, extension of captureprobes, sequencing (e.g., of a cleaved extended capture probe and/or acDNA molecule complementary to an extended capture probe), sequencing onthe array (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in Section (II)(g) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663. Some quality control measuresare described in Section (II)(h) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin Section (II)(c) of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Exemplary features and geometricattributes of an array can be found in Sections (II)(d)(i),(II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells) comprising capture probes). As usedherein, “contact,” “contacted,” and/or “contacting,” a biological samplewith a substrate refers to any contact (e.g., direct or indirect) suchthat capture probes can interact (e.g., bind covalently ornon-covalently (e.g., hybridize)) with analytes from the biologicalsample. Capture can be achieved actively (e.g., using electrophoresis)or passively (e.g., using diffusion). Analyte capture is furtherdescribed in Section (II)(e) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in Section (III) of WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a connected probe (e.g., a ligation product). In someinstances, the two oligonucleotides hybridize to sequences that are notadjacent to one another. For example, hybridization of the twooligonucleotides creates a gap between the hybridized oligonucleotides.In some instances, a polymerase (e.g., a DNA polymerase) can extend oneof the oligonucleotides prior to ligation. After ligation, the connectedprobe (e.g., a ligation product) is released from the analyte. In someinstances, the connected probe (e.g., a ligation product) is releasedusing an endonuclease (e.g., RNAse H). The released connected probe(e.g., a ligation product) can then be captured by capture probes (e.g.,instead of direct capture of an analyte) on an array, optionallyamplified, and sequenced, thus determining the location and optionallythe abundance of the analyte in the biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in the ExemplaryEmbodiments section of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See, for example, the Exemplary embodimentstarting with “In some non-limiting examples of the workflows describedherein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S.Patent Application Publication No. 2020/0277663. See also, e.g., theVisium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C,dated June 2020), and/or the Visium Spatial Tissue Optimization ReagentKits User Guide (e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described inSections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. PatentApplication Publication No. 2020/0277663, or any of one or more of thedevices or methods described in Sections Control Slide for Imaging,Methods of Using Control Slides and Substrates for, Systems of UsingControl Slides and Substrates for Imaging, and/or Sample and ArrayAlignment Devices and Methods, Informational labels of WO 2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in PCTApplication No. 2020/061064 and/or U.S. patent application Ser. No.16/951,854.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2020/061108and/or U.S. patent application Ser. No. 16/951,864.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in the Substrate AttributesSection, Control Slide for Imaging Section of WO 2020/123320, PCTApplication No. 2020/061066, and/or U.S. patent application Ser. No.16/951,843. Fiducial markers can be used as a point of reference ormeasurement scale for alignment (e.g., to align a sample and an array,to align two substrates, to determine a location of a sample or array ona substrate relative to a fiducial marker) and/or for quantitativemeasurements of sizes and/or distances.

II. Targeted RNA Depletion

Targeted RNA depletion allows for depletion or removal of one or morespecies of undesirable RNA molecules (e.g., ribosomal RNA and/ormitochondrial RNA), thereby reducing the pool and concentration ofundesirable RNA molecules in the sample which could interfere withdesired target detection (e.g., detection of mRNA). To achievedepletion, one or more probes are designed that hybridize to one or moreundesirable RNA molecules. For example, in one embodiment, probes can beadministered to a biological sample that selectively hybridize toribosomal RNA (rRNA), thereby reducing the pool and concentration ofrRNA in the sample. In one embodiment, probes can be administered to abiological sample that selectively hybridize to mitochondria RNA(mtRNA), thereby reducing the pool and concentration of mtRNA in thesample. Subsequent or concurrent application of capture probes to thesample can result in improved capture of other types of RNA due to areduction in undesirable RNA (e.g., down-selected RNA) present in thesample.

A non-limiting example of a method for identifying a location of ananalyte (e.g., any of the analytes described herein) in a biologicalsample using targeted RNA depletion includes: (a) contacting thebiological sample with a plurality of undesirable RNA depletion probes(e.g., any of the undesirable RNA depletion probes described herein),wherein an undesirable RNA depletion probe in the plurality ofundesirable RNA depletion probes is substantially complementary to allor a portion of the sequence of an undesirable RNA molecule (e.g., anyof the undesirable RNA molecules described herein) in the biologicalsample; (b) hybridizing the undesirable RNA depletion probe to theundesirable RNA (e.g., using any of the methods for hybridizing theundesirable RNA depletion probe to the undesirable RNA describedherein); (c) removing the plurality of undesirable RNA depletionprobe-undesirable RNA complexes (e.g., using any of the methods forremoving the plurality of undesirable RNA depletion probe-undesirableRNA complexes described herein); (d) contacting the biological samplewith a substrate (e.g., any of the substrates described herein)comprising a plurality of attached capture probes (e.g., any of thecapture probes described herein), wherein a capture probe of theplurality includes (i) the spatial barcode (e.g., any of the spatialbarcode described herein) and (ii) a capture domain (e.g., any of thecapture domains described herein) that binds specifically to a sequencepresent in the analyte; (e) extending a 3′ end of the capture probeusing the analyte that is specifically bound to the capture domain as atemplate to generate an extended capture probe; and (f) amplifying(e.g., using any of the methods for amplifying described herein) theextended capture probe to produce a nucleic acid.

A non-limiting example of a method for identifying a location of ananalyte in a biological sample using RNA-templated ligation and targetedRNA depletion includes: (a) contacting a biological sample with a firstprobe oligonucleotide, a second probe oligonucleotide, and a pluralityof undesirable RNA depletion probes, wherein the first probeoligonucleotide and the second probe oligonucleotide are substantiallycomplementary to adjacent sequences of the analyte, wherein the secondprobe oligonucleotide includes a capture probe binding domain that iscapable of binding to a capture domain of a capture probe, and whereinan undesirable RNA depletion probe of the plurality of undesirable RNAdepletion probes is substantially complementary to a sequence of anundesirable RNA molecule in the biological sample (b) hybridizing thefirst probe oligonucleotide and the second probe oligonucleotide to theanalyte; (c) hybridizing the undesirable RNA depletion probe to theundesirable RNA molecule; (d) ligating the first probe oligonucleotideand the second probe oligonucleotide, thereby creating a ligated probethat is substantially complementary to the analyte; (e) removing theplurality of undesirable RNA depletion probe-undesirable RNA complexesand releasing the ligated probe from the analyte; (f) hybridizing thecapture probe binding domain of the ligated probe to a capture domain ofa capture probe that is affixed to the substrate; and (g) determining(i) all or a part of the sequence of the ligated probe specificallybound to the capture domain, or a complement thereof, and (ii) all or apart of the sequence of the spatial barcode, or a complement thereof,and using the determined sequence of (i) and (ii) to identify thelocation of the analyte in the biological sample.

A non-limiting example of the methods described herein using undesirableRNA depletion probes is shown in FIG. 7A biological sample is contactedwith undesirable RNA depletion probes 701 (e.g., ribosomal depletionprobes) where the undesirable RNA depletion probes hybridize 703 to anundesirable RNA molecule 702 (e.g., rRNA). The RNA depletion probes canbe ligated together, or not ligated together. The undesirable RNA thatis bound to the undesirable RNA depletion probe are digestedenzymatically 704 using RNAse H 705. Treatment with RNAse H results indigested undesirable RNA 706. In some embodiments where the RNAdepletion probes are combined with RNA-template ligation, the methoddescribed in FIG. 7 can happen prior to or concurrent with RTL probe(e.g., RHS and LHS probes) hybridization and ligation reaction with thetarget mRNA. The RNase H digestion of the RNA of the RNA:DNA hybrids ofthe RNA depletion method can happened concurrent with that for the mRNAof the target mRNA:DNA probe hybrids created for the RNA templatedligation reaction. In some embodiments, the methods described in FIG. 7can also be performed in any spatial analysis methodology which wouldbenefit from the removal of undesirable RNA species. For example, RNAdepletion as described herein could also be used in conjunction with thedirect capture of a mRNA by the capture probe.

Upon depletion of the undesirable RNA, the sample will contain anenriched population of the RNA target of interest (e.g., an mRNAtarget). In some embodiments, the undesirable RNA comprises less than20%, 19%, 18%, 17%, 16% 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or 1%, or any range therein, of the total RNA in the sampleafter depletion of the undesirable RNA (i.e., less than 20%, 19%, 18%,17%, 16% 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or1%, or any range therein compared to a sample that undergoes nodepletion step). Consequently, the enriched population of the RNA targetof interest may comprise at least 99%, 98%, 97%, 96%, 95%, 94%, 93%,92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%, orany range therein, of the total RNA in the sample.

(a) Undesirable RNA Molecule(s)

As used herein, the term “undesirable RNA molecule”, or “undesirableRNA”, refers to an undesired RNA that is the target for depletion fromthe biological sample. In some embodiments, examples of the undesirableRNA include, but are not limited to, messenger RNA (mRNA), ribosomal RNA(rRNA), mitochondrial RNA (mtRNA), transfer RNA (tRNA), microRNA(miRNA), and viral RNA. In some embodiments, the undesirable RNA can bea transcript (e.g., present in a tissue section).

In some embodiments, the undesirable RNA molecule includes 5.8Sribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), asmall nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA),tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA), ormitochondrial RNA (mtRNA). In some embodiments, the undesirable RNAmolecule includes an RNA molecule that is added (e.g., transfected) intoa sample (e.g., a small interfering RNA (siRNA)). The undesirable RNAcan be double-stranded RNA or single-stranded RNA. In embodiments wherethe undesirable RNA is double-stranded it is processed as asingle-stranded RNA prior to depletion. In some embodiments, theundesirable RNA can be circular RNA. In some embodiments, theundesirable RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA). Insome embodiments, the undesirable RNA is from E. coli.

In some embodiments, the undesirable RNA molecule is rRNA. In someembodiments, the rRNA is eukaryotic rRNA. In some embodiments, the rRNAis cytoplasmic rRNA. In some embodiments, the rRNA is mitochondrialrRNA. Cytoplasmic rRNAs include, for example, 28S, 5.8S, 5S and 18SrRNAs. Mitochondrial rRNAs include, for example, 12S and 16S rRNAs. TherRNA may also be prokaryotic rRNA, which includes, for example, 5S, 16S,and 23S rRNA. The sequences for rRNAs are well known to those skilled inthe art and can be readily found in sequence databases such as GenBankor may be found in the literature. For example, the sequence for thehuman 18S rRNA can be found in GenBank as Accession No. M10098 and thehuman 28S rRNA as Accession No. M11167.

In some embodiments, the undesirable RNA molecule is mitochondrial RNA.Mitochondrial RNAs include, for example, 12S rRNA (encoded by MT-RNR1),and 16S rRNA (encoded by MT-RNR2), RNAs encoding electron transportchain proteins (e.g., NADH dehydrogenase, coenzyme Q-cytochrome creductase/cytochrome b, cytochrome c oxidase, ATP synthase, or humanin),and tRNAs (encoded by MT-TA, MT-TR, MT-TN, MT-TD, MT-TC, MT-TE, MT-TQ,MT-TG, MT-TH, MT-TI, MT-TL1, MT-TL2, MT-TK, MT-TM, MT-TF, MT-TP, MT-TS1,MT-TS2, MT-TT, MT-TW, MT-TY, or MT-TV).

In some embodiments, the undesirable RNA is transfer RNA (tRNA). In someembodiments, the undesirable RNA may be a particular mRNA. For example,it may be desirable to remove cellular transcripts that are usuallypresent in abundance. Thus, the undesirable mRNA may include, but is notlimited to, ACTB, GAPDH, and TUBB. Other sequences for tRNA and specificmRNA are well known to those skilled in the art and can be readily foundin sequence databases such as GenBank or may be found in the literature.

In some embodiments, mRNA is not targeted for depletion by undesirableRNA probes. In some embodiments, one or more undesirable RNA depletionprobes do not have a poly-dT that will hybridize to the poly-A tail ofeukaryotic mRNA. In yet another particular embodiment, the undesirableRNA depletion probe targets and specifically hybridizes to human 18S orhuman 28S rRNA. Examples of the sequence of undesirable RNA depletionprobes targeting the full length sequence of human 18S and human 28SrRNA are illustrated in, e.g., US Appl. Publ. No. 2011/0111409 A1, whichis incorporated herein by reference.

In some embodiments, the one or more undesirable RNA molecules is asingle species of RNA. For example, in some embodiments, the one or moreundesirable RNA molecule hybridizes only ribosomal RNA molecules. Insome embodiments, the one or more undesirable RNA molecule hybridizesonly mitochondrial RNA molecules. In some embodiments, the undesirableRNA molecule can be a combination of two or more species of RNA. In someembodiments, the undesirable RNA molecule is an RNA fragment of one ofthe undesirable RNA molecules described herein. In some embodiments, theundesirable RNA molecule is a full length RNA molecule of one of theundesirable RNA molecules described herein.

(b) Design of Undesirable RNA Depletion Probes

In some embodiments, the one or more undesirable RNA depletion probes isa DNA probe. In some embodiments, the DNA probe includes asingle-stranded DNA oligonucleotide having a sequence partially orcompletely complementary to an undesirable RNA and specificallyhybridizes to the undesirable RNA. In some embodiments, the one or moreundesirable RNA depletion probes are at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% complementary to one or more undesirable RNAmolecules. In some embodiments, the one or more undesirable RNAdepletion probes is 100% (i.e., completely) complementary to one or moreundesirable RNA molecules.

In some embodiments, probes used herein have been described in Morlan etal., PLoS One. 2012; 7(8):e42882, which is incorporated by reference inits entirety. In some embodiments, probes used herein have beendescribed in U.S. Appl. Publ. No. 2011/0111409, which is incorporated byreference in its entirety. In some embodiments, probes used herein havebeen described in Adiconis et al., Nat Methods. 2013 July; 10(7):623-9,which is incorporated by reference in its entirety.

The DNA probe can be produced by techniques known in the art. Forexample, in some embodiments, a DNA probe is produced by chemicalsynthesis, by in vitro expression from recombinant nucleic acidmolecules, or by in vivo expression from recombinant nucleic acidmolecules. The undesirable RNA depletion probe may also be produced byamplification of the undesirable RNA, e.g., RT-PCR, asymmetric PCR, orrolling circle amplification.

In some embodiments, the methods of targeted RNA depletion as disclosedherein include multiple undesirable RNA depletion probes. In someembodiments, the undesirable RNA depletion probes include sequences thatare complementary or substantially complementary to one or moreundesirable RNA molecules. Methods provided herein may be applied to asingle undesirable RNA molecule or a plurality of undesirable RNAmolecules.

In some embodiments, the undesirable RNA depletion probe is about 20,about 21, about 22, about 23, about 24, about 25, about 26, about 27,about 28, about 29, about 30, about 31, about 32, about 33, about 34,about 35, about 36, about 37, about 38, about 39, about 40, about 40,about 41, about 42, about 43, about 44, about 45, about 46, about 47,about 48, about 49, about 50, about 51, about 52 about 53, about 54,about 55, about 56, about 57, about 58, about 59, about 60, about 61,about 62, about 63, about 64, about 65, about 66, about 67, about 68,about 69, about 70, about 71, about 72, about 73, about 74, about 75,about 76, about 77, about 78, about 79, about 80, about 81, about 82,about 83, about 84, about 85, about 86, about 87, about 88, about 89,about 90, about 91, about 92, about 93, about 94, about 95, about 96,about 97, about 98, about 99, about 100, about 200, about 300, about400, about 500, about 600, about 700, about 800, about 900, about 1000,about 1500, about 2000, about 3000, about 4000, or about 5000nucleotides in length.

In some embodiments, a single undesirable RNA depletion probe spans theentire length of the undesirable RNA. In some embodiments, theundesirable RNA depletion probe has regions that are not complementaryto un undesirable RNA, so long as such sequences do not substantiallyaffect specific hybridization of the undesirable RNA depletion probe tothe undesirable RNA. In some embodiments, the depletion probes are notcontiguous, such that while they may collectively hybridize across alength of the undesirable RNA there may exist gaps between theindividual depletion probes. For example, in some embodiments, the RNAdepletion probes that target an undesirable RNA are spaced at least one,at least two, at least 5, at least 10, at least 20, at least 30, atleast 50, at least 60 nucleotides apart along the length of theundesirable RNA. As such, there may be a plurality of RNA depletionprobes that will hybridize adjacent to, or non-contiguous to, each otheralong the length, or partially along the length, of the undesirable RNAmolecule.

In some embodiments, the undesirable RNA depletion probe is associatedwith (e.g., conjugated to) a detectable label, an optical label, and ora label as described herein. In some instances, the detectable label isa radioisotope, a fluorescent or chemiluminescent moiety, with an enzymeor ligand, which can be used for detection or confirmation that theprobe has hybridized to the target sequence. The detectable label can bedirectly detectable by itself (e.g., radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, can be indirectlydetectable, e.g., by catalyzing chemical alterations of a chemicalsubstrate compound or composition, which chemical substrate compound orcomposition is directly detectable. The detectable label can bequalitatively detected (e.g., optically or spectrally), or it can bequantified using methods known in the art and/or disclosed herein.

In some embodiments, the methods provided herein include a pool of twoor more undesirable RNA depletion probes. In some embodiments, the poolof undesirable RNA depletion probes include about 100 nM, about 200 nM,about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM,about 800 nM, about 900 nM, or about 1000 nM of each RNA depletionprobe. In some embodiments, the pool of undesirable RNA depletion probesinclude about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM,about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM, or more,of each RNA depletion probe. In some embodiments, the concentration ofan RNA depletion probe in a pool of RNA depletion probes depends on therelative abundance of the undesirable RNA targeted by the specific RNAdepletion probe. For example, ribosomal transcripts 18S and 28S can behighly abundant in a tissue sample. In this case, RNA depletion probestargeting 18S and/or 28S can be present in the pool of undesirable RNAdepletion probes at a higher concentration that other RNA depletionprobes present in the pool.

In some embodiments, an RNA depletion probe includes a nucleic acidsequence of any one of SEQ ID NOs: 1-195. In some embodiments, a pool ofRNA depletion probes includes two or more probes each having a nucleicacid sequence selected from any one of SEQ ID NOs: 1-195.

(c) Hybridization of Undesirable RNA Depletion Probe to the UndesirableRNA Molecule

In some embodiments, one or more undesirable RNA depletion probeshybridize to an undesirable RNA. In some embodiments, one or moreundesirable RNA depletion probes hybridize to one or more portions ofthe sequence of the undesirable RNA molecule. In some embodiments, oneor more undesirable RNA depletion probes hybridize to the completesequence of the undesirable RNA molecule. Hybridization can occur at anundesirable RNA having a sequence that is 100% complementary to theprobe oligonucleotide(s). In some embodiments, hybridization can occurat a target having a sequence that is at least (e.g., at least about)80%, at least (e.g., at least about) 85%, at least (e.g., at leastabout) 90%, at least (e.g., at least about) 95%, at least (e.g., atleast about) 96%, at least (e.g., at least about) 97%, at least (e.g.,at least about) 98%, or at least (e.g., at least about) 99%complementary to the probe oligonucleotide(s).

In some embodiments, the undesirable RNA depletion probe may becomplementary to all or part of an undesirable RNA sequence andtherefore, there may be more than one undesirable RNA probe thatspecifically hybridizes to the undesirable RNA. For example, there maybe at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more undesirable RNAdepletion probes that specifically hybridize to an undesirable RNA. Insome embodiments, the undesirable RNA has a tertiary structure and theundesirable RNA depletion probe can be complementary to an exposedportion of the undesirable RNA sequence.

In some embodiments, one or more undesirable RNA depletion probes canhybridize to the undesirable RNA such that at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or 100% of the complete sequenceof the undesirable RNA is hybridized by the undesirable RNA depletionprobes.

In some embodiments, at least one undesirable RNA depletion probespecifically hybridizes to substantially the entire full length sequenceof the undesirable RNA. As used herein, “substantially the entire fulllength sequence” refers to less than 100% but at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%, or any range therein, of the full length sequence. Insome embodiments, a multiplicity of undesirable RNA depletion probesspecifically binds to substantially the entire full length sequence ofthe target RNA. In another embodiment, a multiplicity of DNA probesspecifically binds to the entire full length sequence, or portionsthereof, of the target RNA, either adjacent or in a non-contiguousmanner.

In some embodiments, the undesirable RNA depletion probe specificallyhybridizes to the undesirable RNA molecule and creates a RNA:DNA hybrid.As used herein, “specifically hybridizes” refers to a state where aspecific DNA probe is able to hybridize with a target RNA, for example,rRNA, over other nucleic acids present in a nucleic acid sample. In someinstances, the DNA probe is first denatured into single-stranded DNA bymethods known in the art, for example, by heating or under alkalineconditions, and then hybridized to the target RNA by methods also knownin the art, for example, by cooling the heated DNA in the presence ofthe target RNA. In some instances, the double-stranded DNA probe isheated to achieve denaturation to a single strand prior to being addedto the biological sample. In some instances, the DNA probe is producedas a single-stranded DNA molecule, in which case no denaturation wouldbe required. The condition under which a DNA probe specificallyhybridizes with an RNA are well known to those of ordinary skill in theart and it will be appreciated that these conditions may vary dependingupon factors including the GC content and length of the probe, thehybridization temperature, the composition of the hybridization reagentor solution, and the degree of hybridization specificity sought.

In some embodiments, the RNA:DNA hybrid is then depleted from thenucleic acid sample. For example, in some embodiments, a ribonuclease(RNase) that specifically targets RNA:DNA hybrids is used to deplete theRNA:DNA hybrid. In some embodiments, RNAse H is used to specificallyhydrolyze the RNA in the RNA:DNA hybrid so that the RNA becomesdegraded. The remaining DNA is then available to hybridize with anotherundesirable RNA sequence.

In some instances, after the RNA:DNA hybrid is created, no further stepsare taken to remove the hybrid (e.g., ribonuclease digestion asdescribed below does not occur). Thus, in some instances, hybridizationserves to “block” (e.g., inhibit binding of) single-stranded undesirableRNA molecules (e.g., rRNA) from associating with probe sequences thattarget e.g., poly(A) tails or other targets of interest. Accordingly, insome aspects, spatial detection methods disclosed herein occur in thepresence of the RNA:DNA hybrid. In instances where the RNA:DNA hybrid iscreated but not removed, detection of RNA molecules of interest isincreased relative to a setting in which no hybrid is created. In someinstances, detection of target RNA molecules of interest is increased byabout 5%, by about 10%, by about 15%, by about 20%, by about 25%, byabout 30%, by about 35%, by about 40%, by about 45%, by about 50%, byabout 55%, by about 60%, by about 65%, by about 70%, by about 75%, byabout 80%, by about 85%, by about 90%, by about 95%, by about 1.5-fold,by about 2.0-fold, by about 2.5-fold, by about 3.0-fold, by about3.5-fold, by about 4.0-fold, by about 4.5-fold, by about 5.0-fold, byabout 6-fold, by about 7-fold, by about 8-fold, by about 9-fold, byabout 10-fold, or more compared to a setting in which no hybrid iscreated.

(d) Removing the Plurality of Undesirable RNA DepletionProbe-Undesirable RNA Complexes.

(i) Ribonuclease Digestion

In some embodiments, the undesirable RNA depletion probe-undesirable RNAcomplex is removed. In some embodiments, the removing step includes theaddition of RNAse H. In some embodiments, the removing step includescontacting the undesirable RNA depletion probe with a ribonuclease(e.g., RNAse H). In some embodiments, the ribonuclease is anendoribonuclease. In some embodiments (e.g., in the setting ofRNA-templated ligation), an endoribonuclease also is used to release theprobe from the analyte. In some embodiments, the endoribonuclease is oneor more of RNase H, RNase A, RNase C, or RNase I, or any combinationsthereof. In some embodiments, the endoribonuclease is RNase H. In someembodiments, the RNase H is RNase H1, RNase H2, or any combinationsthereof. In some embodiments, the RNAse H is a thermostable RNAse H.Thermostable RNAse H may be obtained commercially, including, forexample, Hybridase™ (Lucigen, Middleton, WI).

In some embodiments, the RNAse H degrades the RNA from a RNA:DNA hybridat a temperature range of between 32° C. and 95° C. (e.g., using athermostable RNAse H). In some embodiments, the RNAse H degrades the RNAfrom a RNA:DNA hybrid at a temperature range of between 32° C. and 60°C. In some embodiments, the RNAse H degrades the RNA from a RNA:DNAhybrid at a temperature range of between 37° C. and 60° C. In yetanother embodiment, RNAse H degrades the RNA from a RNA:DNA hybrid at atemperature of about 32° C., about 33° C., about 34° C., about 35° C.,about 36° C., about 37° C., about 38° C., about 39° C., about 40° C.,about 41° C., about 42° C., about 43° C., about 44° C., about 45° C.,about 46° C., about 47° C., about 48° C., about 49° C., about 50° C.,about 51° C., about 52° C., about 53° C., about 54° C., about 55° C.,about 56° C., about 57° C., about 58° C., about 59° C., about 60° C.,about 61° C., about 62° C., about 63° C. or about 64° C.

In some embodiments, the hybridization step and RNA degradation withRNAse H is repeated more than once. In some instances, the hybridizationstep and RNA degradation step is repeated at least twice, at least threetimes, at least four times, at least five times, or more. In someinstances, a wash step is performed between each step using methods andsolutions (e.g., PBS, PBST) disclosed herein and known in the art.

In some embodiments, the ribonuclease can be inactivated by apermeabilization agent, for example concurrent inactivation andpermeabilization of a biological sample. In some instances, thepermeabilization agent is one or more of an organic solvent, across-linking agent, a detergent, and an enzyme known in the art. Insome instances, the permeabilization agent is an endopeptidase orprotease. In some instances, the endopeptidase is pepsin. In someinstances, the endopeptidase is proteinase K. In some instances, theribonuclease is heat inactivated. For example, in some instances, theribonuclease (e.g., other than thermostable RNAse H) is heat inactivatedat 65° C.

In some embodiments, DNA probes that have not hybridized with targetundesirable RNA, or probes that have been released following RNase Hdegradation of the RNA from the RNA:DNA hybrid, can be removed atvarious stages of RNA isolation by DNA degrading enzymes or othertechniques well known in the art. In some embodiments, the DNA degradingenzyme is an exonuclease that digests DNA from in a 5′ to 3′ direction.In some embodiments, the DNA degrading enzyme does not digest thecapture probes attached on the substrate. In some embodiments, the DNAdegrading enzyme is a RecJ exonuclease. A RecJ exonuclease degradessingle-stranded DNA (ssDNA) in the 5′-3′ direction and can participatein homologous recombination and mismatch repair. In some instances, theRecJ exonuclease is isolated from Escherichia coli. In some embodiments,DNA degrading enzyme can be inactivated by a permeabilization agentdisclosed herein.

(ii) Removal of Undesirable RNA-Depletion Probe Complex

In some embodiments, the DNA:RNA complex that includes an undesirableRNA depletion probe and an undesirable RNA is removed using methodsother than adding RNAse H. In some instances, the undesirable RNAdepletion probe includes a capture moiety. As disclosed herein, acapture moiety of the undesirable RNA depletion probe is affixed to(e.g., conjugated to) the nucleic acid sequence of the undesirable RNAdepletion probe. In some embodiments, the undesirable RNA depletionprobe includes one or more capture moieties. In some embodiments, thecapture moiety includes a label as described herein. In someembodiments, the label is used to identify and remove an undesirable RNAdepletion probe, whether they are hybridized to undesirable RNAmolecules or not. In some instances, using the label, the RNA depletionprobe (including undesirable RNA depletion probes complexed withundesirable RNA) can be isolated and removed from the biological sample.In some embodiments, the label is directly associated with (i.e.,conjugated to) the undesirable RNA depletion probe. The detectable labelcan be directly detectable by itself (e.g., radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, can beindirectly detectable, e.g., by catalyzing chemical alterations of achemical substrate compound or composition, which chemical substratecompound or composition is directly detectable. Detectable labels can besuitable for small scale detection and/or suitable for high-throughputscreening. As such, suitable detectable labels include, but are notlimited to, radioisotopes, fluorophores, chemiluminescent compounds,bioluminescent compounds, and dyes.

In some embodiments, the capture moiety includes a small molecule. Insome embodiments, the capture moiety includes a nucleic acid. In someembodiments, the nucleic acid is single-stranded. In some embodiments,the nucleic acid is double-stranded. In some embodiments, the nucleicacid is RNA. In some embodiments, the nucleic acid is DNA. In someembodiments, the capture moiety includes a carbohydrate. In someembodiments, the capture moiety is positioned 5′ to the domain in theundesirable RNA depletion probe. In some embodiments, the capture moietyis position 3′ to the domain in the undesirable RNA depletion probe.

In some embodiments, the capture moiety-binding agent that bindsspecifically to the capture moiety includes a protein. In someembodiments, the protein is an antibody. In some embodiments, thecapture moiety-binding agent that binds specifically to the capturemoiety comprises a nucleic acid. In some embodiments, the capturemoiety-binding agent that binds specifically to the capture moietycomprises a small molecule. In some embodiments, the capturemoiety-binding agent that binds specifically to the capture moiety isattached to a substrate. In some embodiments, the substrate is a bead.In some embodiments, the substrate is a well. In some embodiments, thesubstrate is a slide. In some embodiments, the substrate is a magneticbead, for example a paramagnetic particle, such that the undesirable RNAdepletion probe-undesirable RNA complexes, or the undesirable RNAdepletion probe alone, can be removed magnetically from the biologicalsample, for example by a rare earth magnet or other magnetic devices.

In some embodiments, the capture moiety is biotin. In some embodiments,a biotin molecule is directly associated with (i.e., conjugated to) theundesirable RNA depletion probe at the 3′ end. In some embodiments, abiotin molecule is directly associated with (i.e., conjugated to) theundesirable RNA depletion probe at the 5′ end. In some embodiments, thebiotin molecule can be associated to (e.g., conjugated to) an avidinmolecule, allowing pulldown of the undesirable RNA depletionprobe-undesirable RNA complexes, or the undesirable RNA depletion probe.In some embodiments, and as disclosed below, the biotin molecule can beassociated to (e.g., conjugated to) a streptavidin molecule, such thatthe undesirable RNA depletion probe-undesirable RNA complexes, or theundesirable RNA depletion probe conjugated to a biotin molecule can becaptured by streptavidin or avidin and depleted from the biologicalsample.

(e) In Situ Spatial RNA-Templated Ligation (RTL) Using Targeted RNADepletion

In some instances, the undesirable RNA depletion probe is used in thesetting of (e.g., concurrently with) in situ spatial RNA-templatedligation (RTL). In the setting of RTL, removal of undesirable RNA can beachieved concurrently. In some instances, both RTL probeoligonucleotides and undesirable RNA depletion probes can be added atthe same time. After ligation of the RTL probes, an endonuclease such asRNAse H is added to the sample. RNAse H digests both the RNA analyte andthe undesirable RNA. In some instances, at least one of the RTL probesincludes a probe capture sequence such as a poly-A sequence, anoligo-d(T) sequence, or a particular capture sequence (in the setting oftargeted RNA analysis). As a result of this process, undesirable RNAmolecules (e.g., rRNA; mtRNA) are digested and thus are not available tointerfere with downstream applications such as probe capture of thepoly-A sequence or a complement thereof that occurs during spatialarray-based methods disclosed herein.

In one feature of the disclosure, provided are methods for identifying alocation of an analyte in a biological sample, the method comprising (a)contacting a biological sample with a first probe oligonucleotide, asecond probe oligonucleotide, and a plurality of undesirable RNAdepletion probes; (b) hybridizing the first probe oligonucleotide andthe second probe oligonucleotide to the analyte; (c) hybridizing theundesirable RNA depletion probe to the undesirable RNA molecule; (d)ligating the first probe oligonucleotide and the second probeoligonucleotide, thereby creating a ligated probe that is substantiallycomplementary to the analyte; (e) removing the plurality of undesirableRNA depletion probe-undesirable RNA complexes and releasing the ligatedprobe from the analyte; (f) contacting the biological sample with asubstrate, wherein the capture probe is affixed to the substrate,wherein the capture probe comprises a spatial barcode and the capturedomain; (g) allowing the capture probe binding domain of the ligatedprobe to specifically bind to the capture domain; and (h) determining(i) all or a part of the sequence of the ligated probe specificallybound to the capture domain, or a complement thereof, and (ii) all or apart of the sequence of the spatial barcode, or a complement thereof,and using the determined sequence of (i) and (ii) to identify thelocation of the analyte in the biological sample.

In some embodiments, the methods as disclosed herein include hybridizingof one or more probe oligonucleotides (e.g., RTL probes) to a targetanalyte (e.g., RNA; e.g., mRNA) of interest. In some embodiments, themethods include hybridization of 2, 3, 4, or more probeoligonucleotides. In some embodiments, the methods include hybridizationof two probe oligonucleotides. In some embodiments, the probeoligonucleotide includes sequences that are complementary orsubstantially complementary to an analyte. For example, in someembodiments, each probe oligonucleotide includes a sequence that iscomplementary or substantially complementary to an mRNA of interest(e.g., to a portion of the sequence of an mRNA of interest). Methodsprovided herein may be applied to hybridization of two or more probeoligonucleotides to a single nucleic acid molecule. In some embodiments,each target analyte includes a first target region and a second targetregion. In some instances, the methods include providing a plurality offirst probe oligonucleotides and a plurality of second probeoligonucleotides. In some instances, a first probe oligonucleotidehybridizes to a first target region of the nucleic acid. In someinstances, a second probe oligonucleotide hybridizes to a second targetregion of the nucleic acid.

In some instances, a first probe oligonucleotide sequence of a firstprobe oligonucleotide of the plurality of first probe oligonucleotidesmay comprise a first reactive moiety. One or more first probeoligonucleotides of the plurality of first probe oligonucleotides maycomprise the same first probe oligonucleotide sequence and/or the samesecond probe oligonucleotide sequence. The plurality of second probeoligonucleotides may each comprise a third probe oligonucleotidesequence complementary to the sequence of a second target region of anucleic acid molecule of the plurality of nucleic acid molecules. Theplurality of second probe oligonucleotides may further comprise a fourthprobe oligonucleotide sequence. A third probe oligonucleotide sequenceof a second probe oligonucleotide of the plurality of second probeoligonucleotides may comprise a second reactive moiety. One or moreprobe oligonucleotides of the second probe oligonucleotides of theplurality of second probe oligonucleotides may comprise the same thirdprobe oligonucleotide sequence and/or, if present, the same fourth probeoligonucleotide sequence. A first probe oligonucleotide sequence of afirst probe oligonucleotide of the plurality of first probeoligonucleotides may hybridize to first target region of a nucleic acidmolecule of the plurality of nucleic acid molecules. A third probeoligonucleotide sequence of a second probe oligonucleotide of theplurality of second probe oligonucleotides may hybridize to the secondtarget region of a nucleic acid molecule of the plurality of nucleicacid molecules. The first and third probe oligonucleotide sequenceshybridized to the first and second target regions, respectively, of anucleic acid molecule of the plurality of nucleic acid molecules may beadjacent to one another such that a first reactive moiety of the firstprobe oligonucleotide sequence is adjacent to a second reactive moietyof the third probe oligonucleotide sequence. The first and secondreactive moieties of the first and second probe oligonucleotideshybridized to nucleic acid molecules of the plurality of nucleic acidmolecules may react to provide a plurality of probeoligonucleotide-linked nucleic acid molecules.

In some embodiments, one of the probe oligonucleotides includes apoly(A) sequence or a complement thereof. In some instances, the poly(A)sequence or a complement thereof is on the 5′ end of one of the probeoligonucleotides. In some instances, the poly(A) sequence or acomplement thereof is on the 3′ end of one of the probeoligonucleotides. In some embodiments, one probe oligonucleotidesincludes a degenerate or UMI sequence. In some embodiments, the UMIsequence is specific to a particular target or set of targets. In someinstances, the UMI sequence or a complement thereof is on the 5′ end ofone of the probe oligonucleotides. In some instances, the UMI sequenceor a complement thereof is on the 3′ end of one of the probeoligonucleotides.

In some instances, the first and second target regions of a nucleic acidmolecule of the plurality of nucleic acid molecules are adjacent to oneanother. In some embodiments, the first and second probeoligonucleotides bind to complementary sequences on the same transcript.In some embodiments, the complementary sequences to which the firstprobe oligonucleotide and the second probe oligonucleotide bind are 1,2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, about 90, about 95, about 100,about 125, or about 150 nucleotides away from each other. Gaps betweenthe probe oligonucleotides may first be filled prior to ligation, using,for example, dNTPs in combination with a polymerase such as Mupolymerase, DNA polymerase, RNA polymerase, reverse transcriptase, VENTpolymerase, Taq polymerase, and/or any combinations, derivatives, andvariants (e.g., engineered mutants) thereof. In some embodiments, whenthe first and second probe oligonucleotides are separated from eachother by one or more nucleotides, deoxyribonucleotides are used toextend and ligate the first and second probe oligonucleotides.

In some instances, the first probe oligonucleotide and the second probeoligonucleotide hybridize to an analyte on the same transcript. In someinstances, the first probe oligonucleotide and the second probeoligonucleotide hybridize to an analyte on the same exon. In someinstances, the first probe oligonucleotide and the second probeoligonucleotide hybridize to an analyte on different exons. In someinstances, the first probe oligonucleotide and the second probeoligonucleotide hybridize to an analyte that is the result of atranslocation event (e.g., in the setting of cancer). The methodsprovided herein make it possible to identify alternative splicingevents, translocation events, and mutations that change thehybridization rate of one or both probe oligonucleotides (e.g., singlenucleotide polymorphisms, insertions, deletions, point mutations).

In some embodiments, the first and/or second probe as disclosed hereinincludes at least two ribonucleic acid bases at the 3′ end; a functionalsequence; a phosphorylated nucleotide at the 5′ end; and/or a captureprobe binding domain. In some embodiments, the functional sequence is aprimer sequence. The capture probe binding domain is a sequence that iscomplementary to a particular capture domain present in a capture probe.In some embodiments, the capture probe binding domain includes a poly(A)sequence. In some embodiments, the capture probe binding domain includesa poly-uridine sequence, a poly-thymidine sequence, or a combinationthereof. In some embodiments, the capture probe binding domain includesa random sequence (e.g., a random hexamer or octamer). In someembodiments, the capture probe binding domain is complementary to acapture domain in a capture probe that detects a particular target(s) ofinterest. In some embodiments, a capture probe binding domain blockingmoiety that interacts with the capture probe binding domain is provided.In some embodiments, a capture probe binding domain blocking moietyincludes a sequence that is complementary or substantially complementaryto a capture probe binding domain. In some embodiments, a capture probebinding domain blocking moiety prevents the capture probe binding domainfrom binding the capture probe when present. In some embodiments, acapture probe binding domain blocking moiety is removed prior to bindingthe capture probe binding domain (e.g., present in a ligated probe) to acapture probe. In some embodiments, a capture probe binding domainblocking moiety comprises a poly-uridine sequence, a poly-thymidinesequence, or a combination thereof.

In some embodiments, the first probe oligonucleotide hybridizes to ananalyte and a second probe oligonucleotide hybridizes to an analyte inproximity to the first probe oligonucleotide. Hybridization can occur ata target having a sequence that is 100% complementary to the probeoligonucleotide(s). In some embodiments, hybridization can occur at atarget having a sequence that is at least (e.g., at least about) 80%, atleast (e.g., at least about) 85%, at least (e.g., at least about) 90%,at least (e.g., at least about) 95%, at least (e.g., at least about)96%, at least (e.g., at least about) 97%, at least (e.g., at leastabout) 98%, or at least (e.g., at least about) 99% complementary to theprobe oligonucleotide(s). After hybridization, in some embodiments, thefirst probe oligonucleotide is extended. After hybridization, in someembodiments, the second probe oligonucleotide is extended. For example,in some instances a first probe oligonucleotide hybridizes to a targetsequence upstream for a second oligonucleotide probe, whereas in otherinstances a first probe oligonucleotide hybridizes to a target sequencedownstream of a second probe oligonucleotide.

The method disclosed herein include addition of undesirable RNA probesdescribed herein. In some instances, the undesirable RNA probes areadded at the same time as the first probe oligonucleotide and the secondprobe oligonucleotide. In some instances, the undesirable RNA probes areadded before the first probe oligonucleotide and the second probeoligonucleotide. In some instances, the undesirable RNA probes are addedafter the first probe oligonucleotide and the second probeoligonucleotide.

In some embodiments, methods disclosed herein include a wash step afterhybridizing the first and the second probe oligonucleotides and/or theundesirable RNA probes. The wash step removes any unboundoligonucleotides and can be performed using any technique known in theart. In some embodiments, a pre-Hybridization buffer is used to wash thesample. In some embodiments, a phosphate buffer is used. In someembodiments, multiple wash steps are performed to remove unboundoligonucleotides. For example, it is advantageous to decrease the amountof unhybridized probes present in a biological sample as they mayinterfere with downstream applications and methods.

In some embodiments, after hybridization of probe oligonucleotides(e.g., first and the second probe oligonucleotides) to the targetanalyte, the probe oligonucleotides (e.g., the first probeoligonucleotide and the second probe oligonucleotide) are ligatedtogether, creating a single ligated probe that is complementary to thetarget analyte. Ligation can be performed enzymatically or chemically,as described herein. For example, the first and second probeoligonucleotides are hybridized to the first and second target regionsof the analyte, and the probe oligonucleotides are subjected to anucleic acid reaction to ligate them together. For example, the probesmay be subjected to an enzymatic ligation reaction using a ligase (e.g.,T4 RNA ligase (Rnl2), a SplintR ligase, or a T4 DNA ligase). See, e.g.,Zhang L., et al.; Archaeal RNA ligase from Thermoccocus kodakarensis fortemplate dependent ligation RNA Biol. 2017; 14(1): 36-44 for adescription of KOD ligase.

In some embodiments, adenosine triphosphate (ATP) is added during theligation reaction. DNA ligase-catalyzed sealing of nicked DNA substratesis first activated through ATP hydrolysis, resulting in covalentaddition of an AMP group to the enzyme. After binding to a nicked sitein a DNA duplex, the ligase transfers this AMP to the phosphorylated5′-end at the nick, forming a 5′-5′ pyrophosphate bond. Finally, theligase catalyzes an attack on this pyrophosphate bond by the OH group atthe 3′-end of the nick, thereby sealing it, whereafter ligase and AMPare released. If the ligase detaches from the substrate before the 3′attack, e.g., because of premature AMP reloading of the enzyme, then the5′ AMP is left at the 5′-end, blocking further ligation attempts. Insome instances, ATP is added at a concentration of about 1 μM, about 10μM, about 100 μM, about 1000 μM, or about 10000 μM during the ligationreaction.

In some instances, cofactors that aid in joining of the probeoligonuclotides are added during the ligation process. In someinstances, the cofactors include magnesium ions (Mg²⁺). In someinstances, the cofactors include manganese ions (Mn²⁺). In someinstances, Mg²⁺ is added in the form of MgCl₂. In some instances, Mn²⁺is added in the form of MnCl₂. In some instances, the concentration ofMgCl₂ is at about 1 mM, at about 10 mM, at about 100 mM, or at about1000 mM. In some instances, the concentration of MnCl₂ is at about 1 mM,at about 10 mM, at about 100 mM, or at about 1000 mM.

In some embodiments, the probe oligonucleotides (e.g., the first probeoligonucleotide and the second probe oligonucleotide) may each comprisea reactive moiety such that, upon hybridization to the target andexposure to appropriate ligation conditions, the probe oligonucleotidesmay ligate to one another. In some embodiments, probe oligonucleotidesthat include a reactive moiety are ligated chemically. For example, aprobe oligonucleotide capable of hybridizing to a first target region ofa nucleic acid molecule may comprise a first reactive moiety, and aprobe oligonucleotide capable of hybridizing to a second target regionof the nucleic acid molecule may comprise a second reactive moiety. Whenthe first and second probe oligonucleotides are hybridized to the firstand second target regions of the nucleic acid molecule, the first andsecond reactive moieties may be adjacent to one another. A reactivemoiety of a probe may be selected from the non-limiting group consistingof azides, alkynes, nitrones (e.g., 1,3-nitrones), strained alkenes(e.g., trans-cycloalkenes such as cyclooctenes or oxanorbornadiene),tetrazines, tetrazoles, iodides, thioates (e.g., phorphorothioate),acids, amines, and phosphates. For example, the first reactive moiety ofa first probe oligonucleotide may comprise an azide moiety, and a secondreactive moiety of a second probe oligonucleotide may comprise an alkynemoiety. The first and second reactive moieties may react to form alinking moiety. A reaction between the first and second reactivemoieties may be, for example, a cycloaddition reaction such as astrain-promoted azide-alkyne cycloaddition, a copper-catalyzedazide-alkyne cycloaddition, a strain-promoted alkyne-nitronecycloaddition, a Diels-Alder reaction, a [3+2] cycloaddition, a [4+2]cycloaddition, or a [4+1] cycloaddition; a thiol-ene reaction; anucleophilic substation reaction; or another reaction. In someinstances, the ends of the probes are ligated together usingbioorthogonal click chemistry, effectively locking the probes around thetarget. See Rouhanifard et al., Nat Biotechnol. 2018 Nov. 12;10.1038/nbt.4286, which is incorporated by reference in its entirety. Insome cases, reaction between the first and second reactive moieties mayyield a triazole moiety or an isoxazoline moiety. A reaction between thefirst and second reactive moieties may involve subjecting the reactivemoieties to suitable conditions such as a suitable temperature, pH, orpressure and providing one or more reagents or catalysts for thereaction. For example, a reaction between the first and second reactivemoieties may be catalyzed by a copper catalyst, a ruthenium catalyst, ora strained species such as a difluorooctyne, dibenzylcyclooctyne, orbiarylazacyclooctynone. Reaction between a first reactive moiety of afirst probe oligonucleotide hybridized to a first target region of thenucleic acid molecule and a second reactive moiety of a third probeoligonucleotide hybridized to a second target region of the nucleic acidmolecule may link the first probe oligonucleotide and the second probeoligonucleotide to provide a ligated probe. Accordingly, reaction of thefirst and second reactive moieties may comprise a chemical ligationreaction such as a copper-catalyzed 5′ azide to 3′ alkyne “click”chemistry reaction to form a triazole linkage between two probeoligonucleotides. In other non-limiting examples, an iodide moiety maybe chemically ligated to a phosphorothioate moiety to form aphosphorothioate bond, an acid may be ligated to an amine to form anamide bond, and/or a phosphate and amine may be ligated to form aphosphoramidate bond.

In some embodiments, after ligation of the first and second probeoligonucleotides to create a ligated probe, the ligated probe isreleased from the analyte. At this stage of the method, (1) the ligatedprobe is created and is hybridized to the analyte, and (2) theundesirable RNA probe is hybridized to the undesirable RNA. To releasethe ligated probe is released from the analyte, an endoribonuclease isused. An endoribonuclease such as RNAse H specifically cleaves RNA inRNA:DNA hybrids. Thus, not only does RNAse H cleave the hybridization ofthe ligated probe to the analyte (releasing the ligated probe), RNAse Halso cleaves the undesirable RNA. In some embodiments, the ligated probeis released enzymatically. In some embodiments, an endoribonuclease isused to release the probe from the analyte. In some embodiments, theendoribonuclease is one or more of RNase H. In some embodiments, theRNase H is RNase H1 or RNase H2.

In some embodiments, after creating a ligated probe from the probeoligonucleotides (e.g., a first probe oligonucleotide and second probeoligonucleotide), the biological sample is permeabilized. In someembodiments, permeabilization occurs using a protease. In someembodiments, the protease is an endopeptidase. Endopeptidases that canbe used include but are not limited to trypsin, chymotrypsin, elastase,thermolysin, pepsin, clostripan, glutamyl endopeptidase (GluC), ArgC,peptidyl-asp endopeptidase (ApsN), endopeptidase LysC and endopeptidaseLysN. In some embodiments, the endopeptidase is pepsin.

In some embodiments, the ligated probe includes a capture probe bindingdomain, which can hybridize to a capture probe (e.g., a capture probeimmobilized, directly or indirectly, on a substrate). In someembodiments, methods provided herein include contacting a biologicalsample with a substrate, wherein the capture probe is affixed to thesubstrate (e.g., immobilized to the substrate, directly or indirectly).In some embodiments, the capture probe includes a spatial barcode andthe capture domain. In some embodiments, the capture probe bindingdomain of the ligated probe specifically binds to the capture domain.After hybridization of the ligated probe to the capture probe, theligated probe is extended at the 3′ end to make a copy of the additionalcomponents (e.g., the spatial barcode) of the capture probe. In someembodiments, methods of ligated probe capture as provided herein includepermeabilization of the biological sample such that the capture probecan more easily hybridize to the captured ligated probe (i.e., comparedto no permeabilization). In some embodiments, reverse transcription (RT)reagents can be added to permeabilized biological samples. Incubationwith the RT reagents can produce spatially-barcoded full-length cDNAfrom the captured analytes (e.g., polyadenylated mRNA). Second strandreagents (e.g., second strand primers, enzymes) can be added to thebiological sample on the slide to initiate second strand synthesis.

The resulting cDNA can be denatured from the capture probe template andtransferred (e.g., to a clean tube) for amplification, and/or libraryconstruction as described herein. The spatially-barcoded, full-lengthcDNA can be amplified via PCR prior to library construction. The cDNAcan then be enzymatically fragmented and size-selected in order tooptimize the cDNA amplicon size. P5, P7, i7, and i5 can be used assample indexes, and TruSeq Read 2 can be added via End Repair,A-tailing, Adaptor Ligation, and PCR. The cDNA fragments can then besequenced using paired-end sequencing using TruSeq Read 1 and TruSeqRead 2 as sequencing primer sites.

In some embodiments, the biological sample is contacted with theundesirable RNA depletion probes and the RTL probes (e.g., the firstprobe oligonucleotide and the second probe oligonucleotide) atsubstantially the same time. In some embodiments, the biological sampleis contacted with the RTL probes (e.g., the first probe oligonucleotideand the second probe oligonucleotide) after the undesirable RNAdepletion probes.

In some embodiments, the hybridization between the RTL probes (e.g., thefirst probe oligonucleotide and the second probe oligonucleotide) to theanalyte and the hybridization between the undesirable RNA depletionprobes to the undesirable RNA occurs at substantially the same time. Insome embodiments, the hybridization between the RTL probes (e.g., thefirst probe oligonucleotide and the second probe oligonucleotide) to theanalyte occurs after the hybridization between the undesirable RNAdepletion probes to the undesirable RNA occurs substantially the sametime.

In some embodiments, the step of removing the plurality of undesirableRNA depletion probe-undesirable RNA complexes and releasing the ligatedprobe from the analyte occur substantially the same time. In someembodiments, the step of removing the plurality of undesirable RNAdepletion probe-undesirable RNA complexes occurs before releasing theligated probe from the analyte.

In some embodiments, the RTL probes (e.g., the first and second probeoligonucleotides) and the analyte (e.g., the target mRNA) hybridize toform an RNA:DNA hybrid at substantially the same time when theundesirable RNA depletion probes and the undesirable RNA hybridize toform an RNA:DNA hybrid. In some embodiments, a ribonuclease (e.g., RNaseH) digests the RNA strands of the RNA:DNA hybrids, where the RNA strandsinclude the analyte and the undesirable RNA molecule.

Detailed descriptions of targeted RNA capture using RNA-templatedligation (RTL) has been disclosed in U.S. application No. 62/952,736,the entirety of which is incorporated herein by reference.

(f) Targeted Capture of Analytes Using Hybridization of TargetOligonucleotide Probes

In some embodiments, one or more target oligonucleotide probes aredesigned to target and hybridize to a plurality of nucleic acids (e.g.,to prepared spatial libraries; e.g., to prepared cDNA libraries). Inthis instance, before targeting one or more target nucleic acids ofinterest, in some embodiments, the biological sample is first contactedwith the undesirable RNA depletion probes as described herein. In someembodiments, a complex of undesirable RNA depletion probes hybridized toan undesirable RNA molecule is formed. In some embodiments, aribonuclease (e.g., RNase H) digests the RNA strands of the RNA:DNAhybrids, where the RNA strands undesirable RNA molecules (e.g., rRNA).

In some embodiments, disclosed herein are methods of depleting anunwanted RNA in a biological sample and include first contacting thebiological sample with a plurality of undesirable RNA depletion probes,wherein an undesirable RNA depletion probe in the plurality ofundesirable RNA depletion probes is substantially complementary to asequence of an undesirable RNA molecule in the biological sample;hybridizing the undesirable RNA depletion probe to the undesirable RNA;and removing the plurality of undesirable RNA depletionprobe-undesirable RNA complexes. After removal of the undesirable RNAdepletion probe-undesirable RNA complexes, identification of an analytecan be pursued.

In some instances, to create a non-specific library of analytes from asample whose unwanted RNA molecules was depleted, the methods (afterunwanted RNA depletion) include hybridizing the analytes to a pluralityto capture probes, each including a spatial barcode and a capture domainthat binds specifically to a sequence present in the analyte. In someembodiments, the capture probe is extended using the analyte that isspecifically bound to the capture domain as a template to generate anextended capture probe. After template production, the captureprobe/analyte complex can be amplified to produce a cDNA library usingmethods disclosed herein.

After RNA depletion, particular target analytes can be analyzed. Forexample, in one instance disclosed herein, target oligonucleotide probesare part of a “panel” that includes hundreds or thousands ofoligonucleotide probes specific for certain settings. For example, apanel of oligonucleotides can detect analytes dysregulated duringcancer, during immune-dysregulation, during neurological development anddisease progression, or acting in the same pathway. Panels andparticular oligonucleotides are disclosed in U.S. Appl. No. 62/970,066;62/929,686; 62/980,124; and 62/980,116, each of which is incorporated byreference in its entirety.

In some instances, the target oligonucleotide probes hybridize to atarget analyte, and then they are selectively enriched e.g., byamplification and/or pulldown methods disclosed herein. In someembodiments, the target oligonucleotide probe does not include a moietyaffixed to the sequence (i.e., the target oligonucleotide probe is anaked target oligonucleotide probe). In some instances, theoligonucleotide probes are associated with one or more moieties. In someembodiments, the moiety is biotin. In some embodiments, a biotinmolecule is directly associated with (i.e., conjugated to) the targetoligonucleotide probe at the 3′ end. In some embodiments, a biotinmolecule is directly associated with (i.e., conjugated to) the targetoligonucleotide probe at the 5′ end. In some embodiments, and asdisclosed below, the biotin molecule can be associated to (e.g.,conjugated to) an avidin molecule, allowing pulldown of an analyte. Insome embodiments, and as disclosed below, the biotin molecule can beassociated to (e.g., conjugated to) a streptavidin molecule, allowingpulldown of an analyte. After pulldown of the analytes of interest, theresulting analyte can be amplified, creating an enriched library ofanalytes. By “enriched,” it is meant that there are increasedconcentrations of an analyte of interest compared to a sample of thesame library of analytes that has not undergone the pulldown step.

(g) Methods of Targeted RNA Depletion

Provided herein are methods for identifying a location of an analyte(e.g., any of the analyte described herein) in a biological sample thatinclude (a) contacting the biological sample with a substrate comprisinga plurality of attached capture probes, wherein a capture probe of theplurality comprises (i) the spatial barcode and (ii) a capture domainthat binds specifically to a capture probe capture domain; (b)contacting a biological sample with a first probe oligonucleotide, asecond probe oligonucleotide, and a plurality of undesirable RNAdepletion probes (e.g., any of the undesirable RNA depletion probesdescribed herein), wherein the first probe oligonucleotide and thesecond probe oligonucleotide are substantially complementary to adjacentsequences of the analyte, wherein the second probe oligonucleotidecomprises a capture probe binding domain (e.g., any of the capture probebinding domains described herein) that is capable of binding to acapture domain (e.g., any of the capture domains described herein) of acapture probe (e.g., any of the capture probes described herein), andwherein an undesirable RNA depletion probe of the plurality ofundesirable RNA depletion probes is substantially complementary to allor a portion of the sequence of an undesirable RNA molecule (e.g., anyof the undesirable RNA molecules described herein) in the biologicalsample; (c) hybridizing the first probe oligonucleotide and the secondprobe oligonucleotide to the analyte; (d) hybridizing the undesirableRNA depletion probe to the undesirable RNA molecule (e.g., using any ofthe methods for hybridizing the undesirable RNA depletion probe to theundesirable RNA described herein); (e) ligating the first probeoligonucleotide and the second probe oligonucleotide, thereby creating aligated probe (e.g., using any of the methods for ligating describedherein) that is substantially complementary to the analyte; (f) removingthe plurality of undesirable RNA depletion probe-undesirable RNAcomplexes (e.g., using any of the methods for removing the plurality ofundesirable RNA depletion probe-undesirable RNA complexes describedherein) and releasing the ligated probe from the analyte (e.g., usingany of the methods for releasing the ligated probe from the analytedescribed herein); (g) allowing the capture probe binding domain of theligated probe to specifically bind to the capture domain; and (h)determining (i) all or a part of the sequence of the ligated probespecifically bound to the capture domain, or a complement thereof, and(ii) all or a part of the sequence of the spatial barcode, or acomplement thereof, and using the determined sequence of (i) and (ii) toidentify the location of the analyte in the biological sample.

Provided herein are methods for identifying a location of an analyte(e.g., any of the analyte described herein) in a biological sample thatinclude (a) contacting the biological sample with a substrate (e.g., anyof the substrates described herein) comprising a plurality of attachedcapture probes (e.g., any of the capture probes described herein),wherein a capture probe of the plurality comprises (i) the spatialbarcode (e.g., any of the spatial barcode described herein) and (ii) acapture domain (e.g., any of the capture domain described herein) thatbinds specifically to a sequence present in the analyte; (b) contactingthe biological sample with a plurality of undesirable RNA depletionprobes (e.g., any of the undesirable RNA depletion probes describedherein), wherein an undesirable RNA depletion probe in the plurality ofundesirable RNA depletion probes is substantially complementary to allor a portion of the sequence of an undesirable RNA molecule (e.g., anyof the undesirable RNA molecules described herein) in the biologicalsample; (c) hybridizing the undesirable RNA depletion probe to theundesirable RNA (e.g., using any of the methods for hybridizing theundesirable RNA depletion probe to the undesirable RNA describedherein); (d) removing the plurality of undesirable RNA depletionprobe-undesirable RNA complexes (e.g., using any of the methods forremoving the plurality of undesirable RNA depletion probe-undesirableRNA complexes described herein); (e) extending a 3′ end of the captureprobe using the analyte that is specifically bound to the capture domainas a template to generate an extended capture probe; and (f) amplifying(e.g., using any of the methods for amplifying described herein) theextended capture probe to produce a nucleic acid.

Provided herein are methods for identifying a location of an analyte(e.g., any of the analyte described herein) in a biological sample thatinclude (a) contacting the biological sample with a plurality ofundesirable RNA depletion probes (e.g., any of the undesirable RNAdepletion probes described herein), wherein an undesirable RNA depletionprobe in the plurality of undesirable RNA depletion probes issubstantially complementary to all or a portion of the sequence of anundesirable RNA molecule (e.g., any of the undesirable RNA moleculedescribed herein) in the biological sample; (b) hybridizing theundesirable RNA depletion probe to the undesirable RNA (e.g., using anyof the methods for hybridizing the undesirable RNA depletion probe tothe undesirable RNA described herein); (c) removing the plurality ofundesirable RNA depletion probe-undesirable RNA complexes (e.g., usingany of the methods for removing the plurality of undesirable RNAdepletion probe-undesirable RNA complexes described herein); (d)contacting a plurality of nucleic acids with a plurality of targetoligonucleotide probes (e.g., any of the target oligonucleotide probesdescribed herein), wherein: a nucleic acid of the plurality of nucleicacids comprises (i) a spatial barcode (e.g., any of the spatial barcodedescribed herein) or a complement thereof, and (ii) a portion of asequence of an analyte from a biological sample, or a complementthereof; and a target oligonucleotide probe of the plurality of targetoligonucleotide probes comprises: a domain that binds specifically to(i) all or a portion of the spatial barcode or a complement thereof,and/or (ii) all or a portion of the sequence of the analyte from thebiological sample, or a complement thereof, and a molecular tag; (e)enriching a complex of the target oligonucleotide probe specificallybound to the nucleic acid using a substrate comprising an agent (e.g.,any of the agent described herein) that binds specifically to themolecular tag; and (f) determining (i) all or a portion of the sequenceof the spatial barcode or the complement thereof, and (ii) all or aportion of the sequence of the analyte from the biological sample, andusing the determined sequences of (i) and (ii) to identify the locationof the analyte in the biological sample.

In some instances, the undesirable RNA depletion probes are used in asetting where a protein-DNA molecule is used as a target probe. In someinstances, the undesirable RNA depletion probes can be used in any ofthe spatial analysis methods described herein. For example, undesirableRNA depletion probes can hybridize to an undesirable RNA molecule in thepresence of an antibody or antigen binding fragment thereof that isassociated with a nucleic acid molecule, as disclosed herein. In someinstances, the molecule (e.g., a nucleic acid molecule) having a barcode(e.g., a spatial barcode) can be coupled (e.g., associated with;conjugated to) an antibody or antigen binding fragment thereof in amanner that facilitates attachment of the molecule (e.g., a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., a cell; e.g., a surface of a cell) using the antibody orantigen binding fragment thereof. In some instances, the undesirable RNAdepletion probes hybridize to undesirable RNA molecules, disallowing theundesirable RNA molecules from hybridizing to the nucleic acid moleculeof the antibody or antigen binding fragment thereof. In some instances,detection of analytes of interest is increased by about 5%, by about10%, by about 15%, by about 20%, by about 25%, by about 30%, by about35%, by about 40%, by about 45%, by about 50%, by about 55%, by about60%, by about 65%, by about 70%, by about 75%, by about 80%, by about85%, by about 90%, by about 95%, by about 1.5-fold, by about 2.0-fold,by about 2.5-fold, by about 3.0-fold, by about 3.5-fold, by about4.0-fold, by about 4.5-fold, by about 5.0-fold, by about 6-fold, byabout 7-fold, by about 8-fold, by about 9-fold, by about 10-fold, ormore compared to a setting in which no hybrid of undesirable RNAdepletion probe-undesirable RNA is created.

In some instances, the undesirable RNA depletion probe is an RNAmolecule. In some instances, the RNA molecule hybridizes to a DNAmolecule that is conjugated to protein (e.g., an antibody), wherein theantibody binds to a protein of interest. The RNA molecule iscomplementary to the DNA molecule that is conjugated to the protein(e.g., the antibody). In some instances, the following steps areperformed: the antibody-DNA molecule binds to a protein of interest; theRNA molecules (i.e., the RNA depletion probes) hybridize to the DNAmolecule, thereby blocking other nucleic acids from hybridizing to theDNA molecule.

In some instances, the antibody binds to the protein of interest afterhybridizing the RNA molecule to the DNA molecule. In the latter setting,in one embodiment, the RNA molecule is complexed to the antibody-DNAmolecule before the antibody-DNA molecule binds to the protein ofinterest. After the antibody hybridizes to the protein, RNAse H can beadded to cleave the RNA molecule from the DNA molecule such that the DNAmolecule is free to hybridize to any spatial capture array as describedherein.

(h) Pre-Hybridization Methods

(i) Imaging and Staining

Prior to addition of the probes (e.g., undesirable RNA depletion probesand/or RTL probes), in some instances, biological samples can be stainedusing a wide variety of stains and staining techniques. In someinstances, the biological sample is a section on a slide (e.g., a 5 μmsection, a 7 μm section, a 10 μm section, etc.). In some instances, thebiological sample is dried after placement onto a glass slide. In someinstances, the biological sample is dried at 42° C. In some instances,drying occurs for about 1 hour, about 2, hours, about 3 hours, or untilthe sections become transparent. In some instances, the biologicalsample can be dried overnight (e.g., in a desiccator at roomtemperature).

In some embodiments, a sample can be stained using any number ofbiological stains, including but not limited to, acridine orange,Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin,ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine,methyl green, methylene blue, neutral red, Nile blue, Nile red, osmiumtetroxide, propidium iodide, rhodamine, or safranin. In some instances,the methods disclosed herein include imaging the biological sample. Insome instances, imaging the sample occurs prior to deaminating thebiological sample. In some instances, the sample can be stained usingknown staining techniques, including Can-Grunwald, Giemsa, hematoxylinand eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou,Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS)staining techniques. PAS staining is typically performed after formalinor acetone fixation. In some instances, the stain is an H&E stain.

In some embodiments, the biological sample can be stained using adetectable label (e.g., radioisotopes, fluorophores, chemiluminescentcompounds, bioluminescent compounds, and dyes) as described elsewhereherein. In some embodiments, a biological sample is stained using onlyone type of stain or one technique. In some embodiments, stainingincludes biological staining techniques such as H&E staining. In someembodiments, staining includes identifying analytes usingfluorescently-conjugated antibodies. In some embodiments, a biologicalsample is stained using two or more different types of stains, or two ormore different staining techniques. For example, a biological sample canbe prepared by staining and imaging using one technique (e.g., H&Estaining and brightfield imaging), followed by staining and imagingusing another technique (e.g., IHC/IF staining and fluorescencemicroscopy) for the same biological sample.

In some embodiments, biological samples can be destained. Methods ofdestaining or discoloring a biological sample are known in the art, andgenerally depend on the nature of the stain(s) applied to the sample.For example, H&E staining can be destained by washing the sample in HCl,or any other acid (e.g., selenic acid, sulfuric acid, hydroiodic acid,benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid,succinic acid, salicylic acid, tartaric acid, sulfurous acid,trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid,orthophosphoric acid, arsenic acid, selenous acid, chromic acid, citricacid, hydrofluoric acid, nitrous acid, isocyanic acid, formic acid,hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonicacid, hydrogen sulfide, or combinations thereof). In some embodiments,destaining can include 1, 2, 3, 4, 5, or more washes in an acid (e.g.,HCl). In some embodiments, destaining can include adding HCl to adownstream solution (e.g., permeabilization solution). In someembodiments, destaining can include dissolving an enzyme used in thedisclosed methods (e.g., pepsin) in an acid (e.g., HCl) solution. Insome embodiments, after destaining hematoxylin with an acid, otherreagents can be added to the destaining solution to raise the pH for usein other applications. For example, SDS can be added to an aciddestaining solution in order to raise the pH as compared to the aciddestaining solution alone. As another example, in some embodiments, oneor more immunofluorescence stains are applied to the sample via antibodycoupling. Such stains can be removed using techniques such as cleavageof disulfide linkages via treatment with a reducing agent and detergentwashing, chaotropic salt treatment, treatment with antigen retrievalsolution, and treatment with an acidic glycine buffer. Methods formultiplexed staining and destaining are described, for example, inBolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin etal., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem.2009; 57:567-75, and Glass et al., J. Histochem. Cytochem. 2009;57:899-905, the entire contents of each of which are incorporated hereinby reference.

In some embodiments, immunofluorescence or immunohistochemistryprotocols (direct and indirect staining techniques) can be performed asa part of, or in addition to, the exemplary spatial workflows presentedherein. For example, tissue sections can be fixed according to methodsdescribed herein. The biological sample can be transferred to an array(e.g., capture probe array), wherein analytes (e.g., proteins) areprobed using immunofluorescence protocols. For example, the sample canbe rehydrated, blocked, and permeabilized (3×SSC, 2% BSA, 0.1% Triton X,1 U/μl RNAse inhibitor for 10 minutes at 4° C.) before being stainedwith fluorescent primary antibodies (1:100 in 3×SSC, 2% BSA, 0.1% TritonX, 1 U/μl RNAse inhibitor for 30 minutes at 4° C.). The biologicalsample can be washed, coverslipped (in glycerol+1 U/μl RNAse inhibitor),imaged (e.g., using a confocal microscope or other apparatus capable offluorescent detection), washed, and processed according to analytecapture or spatial workflows described herein.

In some instances, a glycerol solution and a cover slip can be added tothe sample. In some instances, the glycerol solution can include acounterstain (e.g., DAPI).

As used herein, an antigen retrieval buffer can improve antibody capturein IF/IHC protocols. An exemplary protocol for antigen retrieval can bepreheating the antigen retrieval buffer (e.g., to 95° C.), immersing thebiological sample in the heated antigen retrieval buffer for apredetermined time, and then removing the biological sample from theantigen retrieval buffer and washing the biological sample.

In some embodiments, optimizing permeabilization can be useful foridentifying intracellular analytes. Permeabilization optimization caninclude selection of permeabilization agents, concentration ofpermeabilization agents, and permeabilization duration. Tissuepermeabilization is discussed elsewhere herein.

In some embodiments, blocking an array and/or a biological sample inpreparation of labeling the biological sample decreases nonspecificbinding of the antibodies to the array and/or biological sample(decreases background). Some embodiments provide for blockingbuffers/blocking solutions that can be applied before and/or duringapplication of the label, wherein the blocking buffer can include ablocking agent, and optionally a surfactant and/or a salt solution. Insome embodiments, a blocking agent can be bovine serum albumin (BSA),serum, gelatin (e.g., fish gelatin), milk (e.g., non-fat dry milk),casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), orpolyvinylpyrrolidone (PVP), biotin blocking reagent, a peroxidaseblocking reagent, levamisole, Carnoy's solution, glycine, lysine, sodiumborohydride, pontamine sky blue, Sudan Black, trypan blue, FITC blockingagent, and/or acetic acid. The blocking buffer/blocking solution can beapplied to the array and/or biological sample prior to and/or duringlabeling (e.g., application of fluorophore-conjugated antibodies) to thebiological sample.

(ii) Preparation of a Sample for Application of Probes

In some instances, the biological sample is deparaffinized.Deparaffinization can be achieved using any method known in the art. Forexample, in some instances, the biological samples is treated with aseries of washes that include xylene and various concentrations ofethanol. In some instances, methods of deparaffinization includetreatment of xylene (e.g., three washes at 5 minutes each). In someinstances, the methods further include treatment with ethanol (e.g.,100% ethanol, two washes 10 minutes each; 95% ethanol, two washes 10minutes each; 70% ethanol, two washes 10 minutes each; 50% ethanol, twowashes 10 minutes each). In some instances, after ethanol washes, thebiological sample can be washed with deionized water (e.g., two washesfor 5 minutes each). It is appreciated that one skilled in the art canadjust these methods to optimize deparaffinization.

In some instances, the biological sample is decrosslinked. In someinstances, the biological sample is decrosslinked in a solutioncontaining TE buffer (comprising Tris and EDTA). In some instances, theTE buffer is basic (e.g., at a pH of about 9). In some instances,decrosslinking occurs at about 50° C. to about 80° C. In some instances,decrosslinking occurs at about 70° C. In some instances, decrosslinkingoccurs for about 1 hour at 70° C. Just prior to decrosslinking, thebiological sample can be treated with an acid (e.g., 0.1M HCl for about1 minute). After the decrosslinking step, the biological sample can bewashed (e.g., with 1×PBST).

In some instances, the methods of preparing a biological sample forprobe application include permeabilizing the sample. In some instances,the biological sample is permeabilized using a phosphate buffer. In someinstances, the phosphate buffer is PBS (e.g., 1×PBS). In some instances,the phosphate buffer is PBST (e.g., 1×PBST). In some instances, thepermeabilization step is performed multiple times (e.g., 3 times at 5minutes each).

In some instances, the methods of preparing a biological sample forprobe application include steps of equilibrating and blocking thebiological sample. In some instances, equilibrating is performed using apre-hybridization (pre-Hyb) buffer. In some instances, the pre-Hybbuffer is RNase-free. In some instances, the pre-Hyb buffer contains nobovine serum albumin (BSA), solutions like Denhardt's, or otherpotentially nuclease-contaminated biological materials.

In some instances, the equilibrating step is performed multiple times(e.g., 2 times at 5 minutes each; 3 times at 5 minutes each). In someinstances, the biological sample is blocked with a blocking buffer. Insome instances, the blocking buffer includes a carrier such as tRNA, forexample yeast tRNA such as from brewer's yeast (e.g., at a finalconcentration of 10-20 μg/mL). In some instances, blocking can beperformed for 5, 10, 15, 20, 25, or 30 minutes.

Any of the foregoing steps can be optimized for performance. Forexample, one can vary the temperature. In some instances, thepre-hybridization methods are performed at room temperature. In someinstances, the pre-hybridization methods are performed at 4° C. (in someinstances, varying the timeframes provided herein).

(i) Hybridizing the Probes

In some embodiments, the methods described herein include hybridizingundesirable RNA depletion probes prior to or contemporaneously withtargeted RNA capture that includes hybridizing a first probeoligonucleotide and a second probe oligonucleotide (e.g., a probe pair).In some instances, the first and second probe oligonucleotides fortargeted RNA capture each include sequences that are substantiallycomplementary to one or more sequences (e.g., one or more targetsequences) of an analyte of interest. In some embodiments, the firstprobe and the second probe bind to complementary sequences that arecompletely adjacent (i.e., no gap of nucleotides) to one another or areon the same transcript.

In some instances, the methods include hybridization of probe sets,wherein the probe pairs are in a medium at a concentration of about 1 toabout 100 nM. In some instances, the concentration of the probe pairs isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, or 500 nM. In some instances, theconcentration of the probe pairs is 5 nM. In some instances, the probesets are diluted in a hybridization (Hyb) buffer. In some instances, theprobe sets are at a concentration of 5 nM in Hyb buffer.

In some instances, probe hybridization (e.g., hybridizing theundesirable RNA depletion probes and/or the first and second probeoligonucleotides) occurs at about 50° C. In some instances, thetemperature of probe hybridization ranges from about 30° C. to about 75°C., from about 35° C. to about 70° C., or from about 40° C. to about 65°C. In some embodiments, the temperature is about 30° C., about 31° C.,about 32° C., about 33° C., about 34° C., about 35° C., about 36° C.,about 37° C., about 38° C., about 39° C., about 40° C., about 41° C.,about 42° C., about 43° C., about 44° C., about 45° C., about 46° C.,about 47° C., about 48° C., about 49° C., about 50° C., about 51° C.,about 52° C., about 53° C., about 54° C., about 55° C., about 56° C.,about 57° C., about 58° C., about 59° C., about 60° C., about 61° C.,about 62° C., about 63° C., about 64° C., about 65° C., about 66° C.,about 67° C., about 68° C., about 69° C., or about 70° C. In someinstances, probe hybridization occurs for about 30 minutes, about 1hour, about 2 hours, about 2.5 hours, about 3 hours, or more. In someinstances, probe hybridization occurs for about 2.5 hours at 50° C.

In some instances, the hybridization buffer includes SSC (e.g., 1×SSC)or SSPE. In some instances, the hybridization buffer includes formamideor ethylene carbonate. In some instances, the hybridization bufferincludes one or more salts, like Mg salt for example MgCl₂, Na salt forexample NaCl, Mn salt for example MnCl₂. In some instances, thehybridization buffer includes Denhardt's solution, dextran sulfate,ficoll, PEG or other hybridization rate accelerators. In some instances,the hybridization buffer includes a carrier such as yeast tRNA, salmonsperm DNA, and/or lambda phage DNA. In some instances, the hybridizationbuffer includes one or more blockers. In some instances, thehybridization buffer includes RNase inhibitor(s). In some instances, thehybridization buffer can include BSA, sequence specific blockers,non-specific blockers, EDTA, RNase inhibitor(s), betaine, TMAC, or DMSO.In some instances, a hybridization buffer can further include detergentssuch as Tween, Triton-X 100, sarkosyl, and SDS. In some instances, thehybridization buffer includes nuclease-free water, DEPC water.

In some embodiments, the complementary sequences to which the firstprobe oligonucleotide and the second probe oligonucleotide bind are 1,2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, about 90, about 95, about 100,about 125, about 150, about 175, about 200, about 250, about 300, about350, about 400, about 450, about 500, about 600, about 700, about 800,about 900, or about 1000 nucleotides away from each other. Gaps betweenthe probe oligonucleotides may first be filled prior to ligation, using,for example, Mu polymerase, DNA polymerase, RNA polymerase, reversetranscriptase, VENT polymerase, Taq polymerase, and/or any combinations,derivatives, and variants (e.g., engineered mutants) thereof. In someembodiments, when the first and second probe oligonucleotides areseparated from each other by one or more nucleotides, nucleotides areligated between the first and second probe oligonucleotides. In someembodiments, when the first and second probe oligonucleotides areseparated from each other by one or more nucleotides,deoxyribonucleotides are ligated between the first and second probeoligonucleotides.

In some instances, after hybridization, the biological sample is washedwith a post-hybridization wash buffer. In some instances, thepost-hybridization wash buffer includes one or more of SSC, yeast tRNA,formamide, ethylene carbonate, and nuclease-free water.

Additional embodiments regarding probe hybridization are furtherprovided.

(i) Hybridizing Temperatures

In some embodiments, the method described utilizes oligonucleotides thatinclude deoxyribonucleic acids (instead of strictly utilizingribonucleotides) at the site of ligation. Utilizing deoxyribonucleicacids in the methods described herein create a more uniform efficiencythat can be readily-controlled and flexible for various applications. Insome embodiments, an undesirable RNA depletion probe includesdeoxyribonucleic acids (instead of strictly utilizing ribonucleotides)at the site of ligation. In some embodiments, a first probeoligonucleotide and/or a second probe oligonucleotide includedeoxyribonucleic acids (instead of strictly utilizing ribonucleotides)at the site of ligation.

In a non-limiting example, the methods disclosed herein includecontacting a biological sample with a plurality of oligonucleotides(e.g., undesirable RNA depletion probes and/or RTL probes) including, anundesirable RNA depletion probe, a first oligonucleotide (e.g., a firstprobe) and a second oligonucleotide (e.g., a second probe), wherein theundesirable RNA depletion probe includes a sequence that issubstantially complementary to at least a portion of an undesirable RNA,wherein the first oligonucleotide (e.g., the first probe) and the secondoligonucleotide (e.g., the second probe) are complementary to a firstsequence present in an analyte and a second sequence present in theanalyte, respectively; hybridizing the undesirable RNA depletion probe,the first oligonucleotide (e.g., the first probe), and the secondoligonucleotide (e.g., the second probe) to the analyte at a firsttemperature; hybridizing the undesirable RNA depletion probe, and thefirst oligonucleotide (e.g., the first probe) and the secondoligonucleotide (e.g., the second probe) to a third oligonucleotide(e.g., a splint oligonucleotide) at a second temperature such that thefirst oligonucleotide (e.g., the first probe) and the secondoligonucleotide (e.g., the second probe) abut each other; ligating thefirst oligonucleotide (e.g., the first probe) to the secondoligonucleotide (e.g., the second probe) to create a ligation product;contacting the biological sample with a substrate, wherein a captureprobe is immobilized on the substrate, wherein the capture probeincludes a spatial barcode and a capture domain; allowing the ligationproduct to specifically bind to the capture domain; and determining (i)all or a part of the sequence of the ligation product specifically boundto the capture domain, or a complement thereof, and (ii) all or a partof the sequence of the spatial barcode, or a complement thereof, andusing the determined sequence of (i) and (ii) to identify the locationof the analyte in the biological sample; wherein the firstoligonucleotide (e.g., the first probe), the second oligonucleotide(e.g., the second probe), and the third oligonucleotide are DNAoligonucleotides, and wherein the first temperature is a highertemperature than the second temperature.

In some embodiments, the undesirable RNA depletion probe, the firstoligonucleotide (e.g., the first probe), and/or the secondoligonucleotide (e.g., the second probe) hybridize to an analyte at afirst temperature. In some embodiments, the first temperature rangesfrom about 50° C. to about 75° C., from about 55° C. to about 70° C., orfrom about 60° C. to about 65° C. In some embodiments, the firsttemperature is about 55° C., about 56° C., about 57° C., about 58° C.,about 59° C., about 60° C., about 61° C., about 62° C., about 63° C.,about 64° C., about 65° C., about 66° C., about 67° C., about 68° C.,about 69° C., or about 70° C.

In some embodiments, after the step of hybridizing the undesirable RNAdepletion probe, first oligonucleotide (e.g., the first probe), and/orthe second oligonucleotide (e.g., the second probe) to the analyte, awash step is performed to remove unbound oligonucleotides (e.g.,probes). The wash step can be performed using any of the wash methodsand solutions described herein.

In some embodiments, after the step of hybridizing the firstoligonucleotide (e.g., the first probe) and the second oligonucleotide(e.g., the second probe) to the analyte, a third oligonucleotide (e.g.,a splint oligonucleotide) is added to the analyte. In some embodiments,the third oligonucleotide is an oligonucleotide. In some embodiments,the third oligonucleotide is a DNA oligonucleotide.

In some embodiments, the third oligonucleotide includes a sequence thatis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%complementary to a portion of the first probe oligonucleotide (e.g., aportion of the first probe that is not hybridized to the analyte (e.g.,an auxiliary sequence)). In some embodiments, the third oligonucleotideincludes a sequence that is 100% complementary to a portion of the firstoligonucleotide (e.g., the first probe). In some embodiments, the thirdoligonucleotide includes a sequence that is at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% complementary to a portion of thesecond probe oligonucleotide (e.g., a portion of the second probe thatis not hybridized to the analyte (e.g., an auxiliary sequence)). In someembodiments, the third oligonucleotide includes a sequence that is 100%complementary to a portion of the second oligonucleotide (e.g., thesecond probe). In some embodiments, the third oligonucleotide hybridizesto the first oligonucleotide (e.g., the first probe) at thecomplementary portion. In some embodiments, the third oligonucleotidehybridizes to the second oligonucleotide (e.g., the second probe) at thecomplementary portion.

In some embodiments, the third oligonucleotide hybridizes to the firstoligonucleotide (e.g., the first probe) and to the secondoligonucleotide (e.g., the second probe) at a second temperature. Insome embodiments, the second temperature is lower than the firsttemperature at which the first and second oligonucleotides (e.g., thefirst and second probes) bind the analyte. In some embodiments, thesecond temperature ranges from about 15° C. to about 35° C., from about20° C. to about 30° C., or from about 25° C. to about 30° C. In someembodiments, the first temperature is about 15° C., about 16° C., about17° C., about 18° C., about 19° C., about 20° C., about 21° C., about22° C., about 23° C., about 24° C., about 25° C., about 26° C., about27° C., about 28° C., about 29° C., about 30° C., about 31° C., about32° C., about 33° C., about 34° C., or about 35° C. Methods including athird, or splint, oligonucleotide have been described in U.S. PatentPub. No. 2019/0055594A1, which is herein incorporated by reference inits entirety.

In some embodiments, after the step of hybridizing the thirdoligonucleotide to the analyte, a wash step is performed to removeunbound third oligonucleotides. The wash step can be performed using anyof the wash methods and solutions described herein. In some embodiments,after the washing step, the first and second oligonucleotides (e.g., thefirst and second probes) are bound to (e.g., hybridized to) the analyte,and the third oligonucleotide is bound to (e.g., hybridized to) thefirst and second oligonucleotides (e.g., at portions of the first andsecond probes that are not bound to the analyte).

In some embodiments, the first oligonucleotide (e.g., the first probe),the second oligonucleotide (e.g., the second probe), and the thirdoligonucleotide are added to the biological sample at the same time.Then, in some embodiments, the temperature is adjusted to the firsttemperature to allow the first oligonucleotide (e.g., the first probe)and the second oligonucleotide (e.g., the second probe) to hybridize tothe analyte in the biological sample. Next, the temperature is adjustedto the second temperature to allow the third oligonucleotide tohybridize to the first oligonucleotide and the second oligonucleotide.

In some embodiments where a third oligonucleotide hybridizes to a firstprobe and a second probe that are hybridized to targets sequences thatare not directly adjacent in the analyte, the third oligonucleotide isextended to fill the gap between the first probe and the second probe.In some instances, a polymerase (e.g., a DNA polymerase) can extend oneof the probes (e.g., the first probe) prior to ligation.

In some embodiments, a ligation step is performed. Ligation can beperformed using any of the methods described herein. In someembodiments, the step includes ligation of the first oligonucleotide(e.g., the first probe) and the second oligonucleotide (e.g., the secondprobe), forming a ligation product. In some embodiments, the thirdoligonucleotide serves as an oligonucleotide splint to facilitateligation of the first oligonucleotide (e.g., the first probe) and thesecond oligonucleotide (e.g., the second probe). In some embodiments,ligation is chemical ligation. In some embodiments, ligation isenzymatic ligation. In some embodiments, the ligase is a T4 RNA ligase(Rnl2), a splintR ligase, a single stranded DNA ligase, or a T4 DNAligase.

(ii) Hybridization Buffer

In some embodiments, an undesirable RNA depletion probe, a first probe,and/or a second probe are hybridized to the analyte in a hybridizationbuffer. In some instances, the hybridization buffer contains formamide.In other instances the hybridization buffer is formamide free. Formamideis not human friendly and it is a known health hazard. Chemically, itcan oxidize over time, thereby impacting reagent shelf life and, mostimportantly, reagent efficacy. As such, the methods described herein caninclude formamide-free buffers, including formamide-free hybridizationbuffer.

In some embodiments, the formamide-free hybridization buffer is asaline-sodium citrate (SSC) hybridization buffer. In some embodiment,the SSC is present in the SSC hybridization buffer from about 1×SSC toabout 6×SSC (e.g., about 1×SSC to about 5×SSC, about 1×SSC to about4×SSC, about 1×SSC to about 3×SSC, about 1×SSC to about 2×SSC, about2×SSC to about 6×SSC, about 2×SSC to about 5×SSC, about 2×SSC to about4×SSC, about 2×SSC to about 3×SSC, about 3×SSC to about 5×SSC, about3×SSC to about 4×SSC, about 4×SSC to about 6×SSC, about 4×SSC to about6×SSC, about 4×SSC to about 5×SSC, or about 5×SSC to about 6×SSC). Insome embodiments, the SSC is present in the SSC hybridization bufferfrom about 2×SSC to about 4×SSC. In some embodiments, SSPE hybridizationbuffer can be used.

In some embodiments, the SSC hybridization buffer comprises a solvent.In some embodiments, the solvent comprises ethylene carbonate instead offormamide (2020, Kalinka et al., Scientia Agricola 78(4):e20190315). Insome embodiments, ethylene carbonate is present in the SSC hybridizationbuffer from about 10% (w/v) to about 25% (w/v) (e.g., about 10% (w/v) toabout 20% (w/v), about 10% (w/v) to about 15% (w/v), about 15% (w/v) toabout 25% (w/v), about 15% (w/v) to about 20% (w/v), or about 20% (w/v)to about 25% (w/v)). In some embodiments, ethylene carbonate is presentin the SSC hybridization buffer from about 15% (w/v) to about 20% (w/v).In some embodiments, ethylene carbonate is present in the SSChybridization buffer at about 10% (w/v), about 11% (w/v), about 12%(w/v), about 13% (w/v), about 14% (w/v), about 15% (w/v), about 16%(w/v), about 17% (w/v), about 18% (w/v), about 19% (w/v), about 20%(w/v), about 21% (w/v), about 22% (w/v), about 23% (w/v), about 24%(w/v), or about 25% (w/v). In some embodiments, ethylene carbonate ispresent in the SSC hybridization buffer at about 13% (w/v).

In some embodiments, the SSC hybridization buffer is at a temperaturefrom about 40° C. to about 60° C. (e.g., about 40° C. to about 55° C.,about 40° C. to about 50° C., about 40° C. to about 45° C., about 45° C.to about 60° C., about 45° C. to about 55° C., about 45° C. to about 50°C., about 50° C. to about 60° C., about 50° C. to about 55° C., or about55° C. to about 60° C.). In some embodiments, the SSC hybridizationbuffer is at temperature from about 45° C. to about 55° C., or any ofthe subranges described herein. In some embodiments, the SSChybridization buffer is at a temperature of about 40° C., about 41° C.,about 42° C., about 43° C., about 44° C., about 45° C., about 46° C.,about 47° C., about 48° C., about 49° C., about 50° C., about 51° C.,about 52° C., about 53° C., about 54° C., about 55° C., about 56° C.,about 57° C., about 58° C., about 59° C., or about 60° C. In someembodiments, the SSC hybridization buffer is at a temperature of about50° C.

In some embodiments, the SSC hybridization buffer further comprises oneor more of a carrier, a crowder, or an additive. Non-limiting examplesof a carrier that can be included in the hybridization buffer include:yeast tRNA, salmon sperm DNA, lambda phage DNA, glycogen, andcholesterol. Non-limiting examples of a molecular crowder that can beincluded in the hybridization buffer include: Ficoll, dextran,Denhardt's solution, and PEG. Non-limiting examples of additives thatcan be included in the hybridization buffer include: binding blockers,RNase inhibitors, Tm adjustors and adjuvants for relaxing secondarynucleic acid structures (e.g., betaine, TMAC, and DMSO). Further, ahybridization buffer can include detergents such as SDS, Tween, Triton-X100, and sarkosyl (e.g., N-Lauroylsarcosine sodium salt). A skilledartisan would understand that a buffer for hybridization of nucleicacids could include many different compounds that could enhance thehybridization reaction.

(j) Washing

In some embodiments, the methods disclosed herein also include a washstep. The wash step removes any unbound probes. Wash steps could beperformed between any of the steps in the methods disclosed herein. Forexample, a wash step can be performed after adding probes (e.g., any ofthe undesirable RNA probes and/or RTL probe pairs described herein) tothe biological sample. As such, free/unbound probes are washed away,leaving only probes that have hybridized to an analyte and/orundesirable RNA (e.g., rRNA). In some instances, multiple (i.e., atleast 2, 3, 4, 5, or more) wash steps occur between the methodsdisclosed herein. Wash steps can be performed at times (e.g., 1, 2, 3,4, or 5 minutes) and temperatures (e.g., room temperature; 4° C. knownin the art and determined by a person of skill in the art.

In some instances, wash steps are performed using a wash buffer. In someinstances, the wash buffer includes SSC (e.g., 1×SSC). In someinstances, the wash buffer includes PBS (e.g., 1×PBS). In someinstances, the wash buffer includes PBST (e.g., 1×PBST). In someinstances, the wash buffer can also include formamide or be formamidefree.

Additional embodiments regarding wash steps are provided herein.

(i) Formamide Free Wash Buffer

In some embodiments, after hybridizing and/or ligating the undesirableRNA depletion probe, one or more unhybridized undesirable RNA depletionprobes are removed from the array. In some embodiments, after ligating afirst probe and a second probe, the one or more unhybridized firstprobes, one or more unhybridized second probes, or both, are removedfrom the array. In some embodiments, after ligating a first probe, asecond probe, and a third oligonucleotide, the one or more unhybridizedfirst probes, one or more unhybridized second probes, or one or morethird oligonucleotides, or all the above, are removed from the array.

In some embodiments, a pre-hybridization buffer is used to wash thesample. In some embodiments, a phosphate buffer is used. In someembodiments, multiple wash steps are performed to remove unboundoligonucleotides.

In some embodiments, removing includes washing the one or moreunhybridized probes (e.g., an undesirable RNA depletion probe, a firstprobe, a second probe, and a third oligonucleotide) from the array in aformamide-free wash buffer.

In some embodiments, the formamide-free wash buffer is an SSC washbuffer. In some embodiments, SSC is present in the SSC wash buffer fromabout 0.01×SSC to about 1×SSC (e.g., about 0.01×SSC to about 0.5×SSC,0.01×SSC to about 0.1×SSC, about 0.01×SSC to about 0.05×SSC, about0.05×SSC to about 1×SSC, about 0.05×SSC to about 0.5×SSC, about 0.05×SSCto about 0.1×SSC, about 0.1×SSC to about 1×SSC, about 0.1×SSC to about0.5×SSC, or about 0.5×SSC to about 1×SSC). In some embodiments, SSC ispresent in the SSC wash buffer at about 0.01×SSC, about 0.02×SSC, about0.03×SSC, about 0.04×SSC, about 0.05×SSC, about 0.06×SSC, about0.07×SSC, about 0.08×SSC, about 0.09×SSC, about 0.1×SSC, about 0.2×SSC,about 0.3×SSC, about 0.4×SSC, about 0.5×SSC, about 0.6×SSC, about0.7×SSC, about 0.8×SSC, about 0.9×SSC, or about 0.1×SSC. In someembodiments, SSC is present in the SSC wash buffer at about 0.1×SSC.

In some embodiments, the SSC wash buffer comprises a detergent. In someembodiments, the detergent comprises sodium dodecyl sulfate (SDS). Insome embodiments, SDS is present in the SSC wash buffer from about 0.01%(v/v) to about 0.5% (v/v) (e.g., about 0.01% (v/v) to about 0.4% (v/v),about 0.01% (v/v) to about 0.3% (v/v), about 0.01% (v/v) to about 0.2%(v/v), about 0.01% (v/v) to about 0.1% (v/v), about 0.05% (v/v) to about0.5% (v/v), about 0.05% (v/v) to about 0.4% (v/v), about 0.05% (v/v) toabout 0.3% (v/v), about 0.05% (v/v) to about 0.2% (v/v), about 0.05%(v/v) to about 0.1% (v/v), about 0.1% (v/v) to about 0.5% (v/v), about0.1% (v/v) to about 0.4% (v/v), about 0.1% (v/v) to about 0.3% (v/v),about 0.1% (v/v) to about 0.2% (v/v), about 0.2% (v/v) to about 0.5%(v/v), about 0.2% (v/v) to about 0.4% (v/v), about 0.2% (v/v) to about0.3% (v/v), about 0.3% (v/v) to about 0.5% (v/v), about 0.3% (v/v) toabout 0.4% (v/v), or about 0.4% (v/v) to about 0.5% (v/v)). In someembodiments, the SDS is present the SSC wash buffer at about 0.01%(v/v), about 0.02% (v/v), about 0.03% (v/v), about 0.04% (v/v), about0.05% (v/v), about 0.06% (v/v), about 0.07% (v/v), about 0.08% (v/v),about 0.09% (v/v), about 0.10% (v/v), about 0.2% (v/v), about 0.3%(v/v), about 0.4% (v/v), or about 0.5% (v/v), In some embodiments, theSDS is present in the SSC wash buffer at about 0.1% (v/v). In someembodiments, sarkosyl may be present in the SSC wash buffer.

In some embodiments, the SSC wash buffer comprises a solvent. In someembodiments, the solvent comprises formamide or ethylene carbonate. Insome embodiments, ethylene carbonate is present in the SSC wash bufferfrom about 10% (w/v) to about 25% (w/v), or any of the subrangesdescribed herein. In some embodiments, ethylene carbonate is present inthe SSC wash buffer from about 15% (w/v) to about 20% (w/v). In someembodiments, ethylene carbonate is present in the SSC wash buffer atabout 16% (w/v).

In some embodiments, the SSC wash buffer is at a temperature from about50° C. to about 70° C. (e.g., about 50° C. to about 65° C., about 50° C.to about 60° C., about 50° C. to about 55° C., about 55° C. to about 70°C., about 55° C. to about 65° C., about 55° C. to about 60° C., about60° C. to about 70° C., about 60° C. to about 65° C., or about 65° C. toabout 70° C.). In some embodiments, the SSC wash buffer is at atemperature from about 55° C. to about 65° C. In some embodiments, theSSC wash buffer is at a temperature about 50° C., about 51° C., about52° C., about 53° C., about 54° C., about 55° C., about 56° C., about57° C., about 58° C., about 59° C., about 60° C., about 61° C., about62° C., about 63° C., about 64° C., about 65° C., about 66° C., about67° C., about 68° C., about 69° C., or about 70° C. In some embodiments,the SSC wash buffer is at a temperature of about 60° C.

In some embodiments, the method includes releasing the ligation product,where releasing is performed after the array is washed to remove the oneor more unhybridized first and second probes.

(k) Ligation

In some embodiments, after hybridization of the probe oligonucleotides(e.g., a first probe, a second probe, and/or a third oligonucleotide) tothe analyte, the probes (e.g., a first probe, a second probe, and/or athird oligonucleotide) can be ligated together, creating a singleligation product that includes one or more sequences that arecomplementary to the analyte. In some embodiments, after hybridizationof the undesirable RNA depletion probes, the undesirable RNA depletionprobes can be ligated together. Ligation can be performed enzymaticallyor chemically, as described herein.

In some instances, the ligation is an enzymatic ligation reaction, usinga ligase (e.g., T4 RNA ligase (Rnl2), a SplintR ligase, a singlestranded DNA ligase, or a T4 DNA ligase). See, e.g., Zhang et al.; RNABiol. 2017; 14(1): 36-44, which is incorporated by reference in itsentirety, for a description of KOD ligase. Following the enzymaticligation reaction, the probes (e.g., a first probe, a second probe,and/or a third oligonucleotide) may be considered ligated.

In some embodiments, a polymerase catalyzes synthesis of a complementarystrand of the ligation product, creating a double-stranded ligationproduct. In some instances, the polymerase is DNA polymerase. In someembodiments, the polymerase has 5′ to 3′ polymerase activity. In someembodiments, the polymerase has 3′ to 5′ exonuclease activity forproofreading. In some embodiments, the polymerase has 5′ to 3′polymerase activity and 3′ to 5′ exonuclease activity for proofreading.

In some embodiments, the probe (e.g., a first probe, a second probe,and/or a third oligonucleotide) may each comprise a reactive moiety suchthat, upon hybridization to the target and exposure to appropriateligation conditions, the probe oligonucleotides may ligate to oneanother. In some embodiments, probe oligonucleotides that include areactive moiety are ligated chemically. For example, a first probecapable of hybridizing to a first target region (e.g., a first targetsequence or a first portion) of a nucleic acid molecule may comprise afirst reactive moiety, and a second probe oligonucleotide capable ofhybridizing to a second target region (e.g., a second target sequence ora second portion) of the nucleic acid molecule may comprise a secondreactive moiety. When the first and second probes are hybridized to thefirst and second target regions (e.g., first and second targetsequences) of the nucleic acid molecule, the first and second reactivemoieties may be adjacent to one another. A reactive moiety of a probemay be selected from the non-limiting group consisting of azides,alkynes, nitrones (e.g., 1,3-nitrones), strained alkenes (e.g.,trans-cycloalkenes such as cyclooctenes or oxanorbornadiene),tetrazines, tetrazoles, iodides, thioates (e.g., phorphorothioate),acids, amines, and phosphates. For example, the first reactive moiety ofa first probe may comprise an azide moiety, and a second reactive moietyof a second probe may comprise an alkyne moiety. The first and secondreactive moieties may react to form a linking moiety. A reaction betweenthe first and second reactive moieties may be, for example, acycloaddition reaction such as a strain-promoted azide-alkynecycloaddition, a copper-catalyzed azide-alkyne cycloaddition, astrain-promoted alkyne-nitrone cycloaddition, a Diels-Alder reaction, a[3+2] cycloaddition, a [4+2] cycloaddition, or a [4+1] cycloaddition; athiol-ene reaction; a nucleophilic substation reaction; or anotherreaction. In some cases, reaction between the first and second reactivemoieties may yield a triazole moiety or an isoxazoline moiety. Areaction between the first and second reactive moieties may involvesubjecting the reactive moieties to suitable conditions such as asuitable temperature, pH, or pressure and providing one or more reagentsor catalysts for the reaction. For example, a reaction between the firstand second reactive moieties may be catalyzed by a copper catalyst, aruthenium catalyst, or a strained species such as a difluorooctyne,dibenzylcyclooctyne, or biarylazacyclooctynone. Reaction between a firstreactive moiety of a first probe hybridized to a first target region(e.g., a first target sequence or first portion) of the nucleic acidmolecule and a second reactive moiety of a third probe oligonucleotidehybridized to a second target region (e.g., a first target sequence or afirst portion) of the nucleic acid molecule may link the first probe andthe second probe to provide a ligated probe. Upon linking, the first andsecond probe may be considered ligated. Accordingly, reaction of thefirst and second reactive moieties may comprise a chemical ligationreaction such as a copper-catalyzed 5′ azide to 3′ alkyne “click”chemistry reaction to form a triazole linkage between two probeoligonucleotides. In other non-limiting examples, an iodide moiety maybe chemically ligated to a phosphorothioate moiety to form aphosphorothioate bond, an acid may be ligated to an amine to form anamide bond, and/or a phosphate and amine may be ligated to form aphosphoramidate bond.

In some instances, ligation is performed in a ligation buffer. Ininstances where probe ligation is performed on diribo-containing probes,the ligation buffer can include T4 RNA Ligase Buffer 2, enzyme (e.g.,RNL2 ligase), and nuclease free water. In instances where probe ligationis performed on DNA probes, the ligation buffer can include Tris-HClpH7.5, MnCl2, ATP, DTT, surrogate fluid (e.g., glycerol), enzyme (e.g.,SplintR ligase), and nuclease-free water.

In some embodiments, the ligation buffer includes additional reagents.In some instances, the ligation buffer includes adenosine triphosphate(ATP) is added during the ligation reaction. DNA ligase-catalyzedsealing of nicked DNA substrates is first activated through ATPhydrolysis, resulting in covalent addition of an AMP group to theenzyme. After binding to a nicked site in a DNA duplex, the ligasetransfers the AMP to the phosphorylated 5′-end at the nick, forming a5′-5′ pyrophosphate bond. Finally, the ligase catalyzes an attack onthis pyrophosphate bond by the OH group at the 3′-end of the nick,thereby sealing it, whereafter ligase and AMP are released. If theligase detaches from the substrate before the 3′ attack, e.g., becauseof premature AMP reloading of the enzyme, then the 5′ AMP is left at the5′-end, blocking further ligation attempts. In some instances, ATP isadded at a concentration of about 1 μM, about 10 μM, about 100 μM, about1000 μM, or about 10000 μM during the ligation reaction.

In some embodiments, cofactors that aid in joining of the probeoligonucleotides are added during the ligation process. In someinstances, the cofactors include magnesium ions (Mg′). In someinstances, the cofactors include manganese ions (Mn′). In someinstances, Mg′ is added in the form of MgCl₂. In some instances, Mn′ isadded in the form of MnCl₂. In some instances, the concentration ofMgCl₂ is at about 1 mM, at about 10 mM, at about 100 mM, or at about1000 mM. In some instances, the concentration of MnCl₂ is at about 1 mM,at about 10 mM, at about 100 mM, or at about 1000 mM.

In some embodiments, the ligation product includes a capture probecapture domain, which can bind to a capture probe (e.g., a capture probeimmobilized, directly or indirectly, on a substrate). In someembodiments, methods provided herein include contacting a biologicalsample with a substrate, wherein the capture probe is affixed to thesubstrate (e.g., immobilized to the substrate, directly or indirectly).In some embodiments, the capture probe capture domain of the ligatedprobe specifically binds to the capture domain.

After ligation, in some instances, the biological sample is washed witha post-ligation wash buffer. In some instances, the post-ligation washbuffer includes one or more of SSC (e.g., 1×SSC), ethylene carbonate orformamide, and nuclease free water. In some instances, the biologicalsample is washed at this stage at about 50° C. to about 70° C. In someinstances, the biological sample is washed at about 60° C.

(i) Ligation Including Pre-Adenylated 5′ Phosphate on Second Probe

Provided herein are methods for determining a location of a targetnucleic acid in a biological sample that include: (a) contacting thebiological sample with a substrate comprising a plurality of captureprobes, where a capture probe of the plurality of capture probescomprises a capture domain and a spatial barcode; (b) hybridizing atarget nucleic acid in the biological sample with a first probe and asecond probe, where the first probe comprises, from 3′ to 5′, a sequencesubstantially complementary to the capture domain and a sequence that issubstantially complementary to a first sequence in the target nucleicacid and has a pre-adenylated phosphate group at its 5′ end; the secondprobe comprises a sequence substantially complementary to a secondsequence in the target nucleic acid; (c) generating a ligation productby ligating a 3′ end of the second probe to the 5′ end of the firstprobe using a ligase that does not require adenosine triphosphate forligase activity; (d) releasing the ligation product from the targetnucleic acid and binding the capture domain of the ligation productspecifically to the capture domain of capture probe; and (e) determining(i) all or a part of a sequence corresponding to the ligation product,or a complement thereof, and (ii) all or a part of a sequencecorresponding to the spatial barcode, or a complement thereof, and usingthe determined sequences of (i) and (ii) to identify the location of thetarget nucleic acid in the biological sample

In some instances, the ligase that does not require adenosinetriphosphate for ligase activity (e.g., thermostable 5′ AppDNA/RNALigase, truncated T4 RNA Ligase 2 (trRnl2), truncated T4 RNA Ligase 2K227Q, truncated T4 RNA Ligase 2 KQ, Chlorella Virus PBCV-1 DNA Ligase,and combinations thereof). See, e.g., Nichols et al., “RNA Ligases,”Curr. Protocol. Molec. Biol. 84(1):3.15.1-.4 (2008); Viollet et al., “T4RNA Ligase 2 Truncated Active Site Mutants: Improved Tools for RNAAnalysis,” BMC Biotechnol. 11: 72 (2011); and Ho et al., “BacteriophageT4 RNA Ligase 2 (gp24.1) Exemplifies a Family of RNA Ligases Found inAll Phylogenetic Domains,” PNAS 99(20):12709-14 (2002), which are herebyincorporated by reference in their entirety for a description of T4 RNALigases and truncated T4 RNA Ligases. Thermostable 5′ AppDNA/RNA Ligaseis an enzyme belonging to the Ligase family that catalyzes the ligationof the 3′ end of ssRNA or ssDNA to a 5′-adenylated ssDNA or5′-adenylated ssRNA. Truncated T4 RNA Ligase 2 is an enzyme belonging tothe Ligase family that catalyzes the ligation of dsRNA nicks and ssRNAto ssRNA. It can also ligate the 3′ end of RNA or DNA to a 5′-pDNA whenannealed to an RNA complement, and the 3′ end of RNA to a 5′-pRNA whenannealed to a DNA complement, with reduced efficiency. Truncated T4 RNALigase 2 K227Q is an enzyme belonging to the Ligase family thatcatalyzes the ligation of the 3′ end of ssRNA to 5′ adenylated ssDNA and5′ adenylated ssRNA. It has a reduction of side products as compared totruncated T4 RNA Ligase 2. Truncated T4 RNA Ligase 2 KQ is an enzymebelonging to the Ligase family that catalyzes the ligation of the 3′ endof ssRNA to 5′ adenylated ssDNA and 5′ adenylated ssRNA. It is apreferred choice for ligation of ssRNA to preadenylated adapters and hasa reduction of side products as compared to truncated T4 RNA Ligase 2.

In some embodiments, the T4 RNA Ligase comprises a K227Q mutation. SeeViollet et al., “T4 RNA Ligase 2 Truncated Active Site Mutants: ImprovedTools for RNA Analysis,” BMC Biotechnol. 11, which is herebyincorporated by reference in its entirety.

In some instances, cofactors that aid in ligation of the first andsecond probe are added during ligation. In some instances, the cofactorsinclude magnesium ions (Mg²⁺). In some instances, the cofactors includemanganese ions (Mn²⁺). In some instances, Mg²⁺ is added in the form ofMgCl₂. In some instances, Mn²⁺ is added in the form of MnCl₂. In someinstances, the concentration of MgCl₂ is at about 1 mM to about 10 mM.In some instances, the concentration of MnCl₂ is at about 1 mM to about10 mM.

In some instances, the ligation occurs at a pH in the range of about 6.5to about 9.0, about 6.5 to about 8.0, or about 7.5 to about 8.0.

In some embodiments, the ligation buffer includes an enzyme storagebuffer. In some embodiments, the enzymes storage buffer includesglycerol. In some embodiments, the ligation buffer is supplemented withglycerol. In some embodiments, the glycerol is present in the ligationbuffer at a total volume of 15% v/v.

(l) Permeabilization and Releasing the Ligation Product

In some embodiments, the methods provided herein include apermeabilizing step. In some embodiments, permeabilization occurs usinga protease. In some embodiments, the protease is an endopeptidase.Endopeptidases that can be used include but are not limited to trypsin,chymotrypsin, elastase, thermolysin, pepsin, clostripan, glutamylendopeptidase (GluC), ArgC, peptidyl-asp endopeptidase (ApsN),endopeptidase LysC and endopeptidase LysN. In some embodiments, theendopeptidase is pepsin. In some embodiments, the biological sample ispermeabilized contemporaneously with or prior to contacting thebiological sample with undesirable RNA depletion probes. In someembodiments, the biological sample is permeabilized after the biologicalsample is contacted with undesirable RNA depletion probes. In someembodiments, the biological sample is permeabilized after the biologicalsample is contacted with undesirable RNA depletion probes but prior tocontacting the array. In some embodiments, the biological sample ispermeabilized after the biological sample is contacted with undesirableRNA depletion probes but prior to contacting a first probeoligonucleotide and a second probe oligonucleotide. In some embodiments,after creating a ligation product (e.g., by ligating a first probe and asecond probe that are hybridized to adjacent sequences in the analyte),the biological sample is permeabilized. In some embodiments, thebiological sample is permeabilized contemporaneously with or prior tocontacting the biological sample with a first probe and a second probe,hybridizing the first probe and the second probe to the analyte,generating a ligation product by ligating the first probe and the secondprobe, and releasing the ligated product from the analyte.

In some embodiments, methods provided herein include permeabilization ofthe biological sample such that the capture probe can more easily bindto the captured ligated probe (i.e., compared to no permeabilization).In some embodiments, reverse transcription (RT) reagents can be added topermeabilized biological samples. Incubation with the RT reagents canproduce spatially-barcoded full-length cDNA from the captured analytes(e.g., polyadenylated mRNA). Second strand reagents (e.g., second strandprimers, enzymes) can be added to the biological sample on the slide toinitiate second strand synthesis.

In some instances, the permeabilization step includes application of apermeabilization buffer to the biological sample. In some instances, thepermeabilization buffer includes a buffer (e.g., Tris pH 7.5), MgCl2,sarkosyl detergent (e.g., sodium lauroyl sarcosinate), enzyme (e.g.,proteinase K), and nuclease free water. In some instances, thepermeabilization step is performed at 37° C. In some instances, thepermeabilization step is performed for about 20 minutes to 2 hours(e.g., about 20 minutes, about 30 minutes, about 40 minutes, about 50minutes, about 1 hour, about 1.5 hours, or about 2 hours). In someinstances, the releasing step is performed for about 40 minutes.

In some embodiments, after generating a ligation product, the ligationproduct is released from the analyte. In some embodiments, a ligationproduct is released from the analyte using an endoribonuclease. In someembodiments, the endoribonuclease is RNase H, RNase A, RNase C, or RNaseI. In some embodiments, the endoribonuclease is RNase H. RNase H is anendoribonuclease that specifically hydrolyzes the phosphodiester bondsof RNA, when hybridized to DNA. RNase H is part of a conserved family ofribonucleases which are present in many different organisms. There aretwo primary classes of RNase H: RNase H1 and RNase H2. Retroviral RNaseH enzymes are similar to the prokaryotic RNase H1. All of these enzymesshare the characteristic that they are able to cleave the RNA componentof an RNA:DNA heteroduplex. In some embodiments, the RNase H is RNaseH1, RNase H2, or RNase H1, or RNase H2. In some embodiments, the RNase Hincludes but is not limited to RNase HII from Pyrococcus furiosus, RNaseHII from Pyrococcus horikoshi, RNase HI from Thermococcus litoralis,RNase HI from Thermus thermophilus, RNAse HI from E. coli, or RNase HIIfrom E. coli.

In some instances, the releasing step is performed using a releasingbuffer. In some instances, the release buffer includes one or more of abuffer (e.g., Tris pH 7.5), enzyme (e.g., RNAse H) and nuclease-freewater. In some instances, the releasing step is performed at 37° C. Insome instances, the releasing step is performed for about 20 minutes to2 hours (e.g., about 20 minutes, about 30 minutes, about 40 minutes,about 50 minutes, about 1 hour, about 1.5 hours, or about 2 hours). Insome instances, the releasing step is performed for about 30 minutes.

In some instances, the releasing step occurs before the permeabilizationstep. In some instances, the releasing step occurs after thepermeabilization step. In some instances, the releasing step occurs atthe same time as the permeabilization step (e.g., in the same buffer).

(m) Biological Samples

Methods disclosed herein can be performed on any type of sample. In someembodiments, the sample is a fresh tissue. In some embodiments, thesample is a frozen sample. In some embodiments, the sample waspreviously frozen. In some embodiments, the sample is a formalin-fixed,paraffin embedded (FFPE) sample.

Subjects from which biological samples can be obtained can be healthy orasymptomatic individuals, individuals that have or are suspected ofhaving a disease (e.g., cancer) or a pre-disposition to a disease,and/or individuals that are in need of therapy or suspected of needingtherapy. In some instances, the biological sample can include one ormore diseased cells. A diseased cell can have altered metabolicproperties, gene expression, protein expression, and/or morphologicfeatures. Examples of diseases include inflammatory disorders, metabolicdisorders, nervous system disorders, and cancer. In some instances, thebiological sample includes cancer or tumor cells. Cancer cells can bederived from solid tumors, hematological malignancies, cell lines, orobtained as circulating tumor cells. In some instances, the biologicalsample is a heterogenous sample. In some instances, the biologicalsample is a heterogenous sample that includes tumor or cancer cellsand/or stromal cells,

In some instances, the cancer is breast cancer. In some instances, thebreast cancer is triple positive breast cancer (TPBC). In someinstances, the breast cancer is triple negative breast cancer (TNBC).

In some instances, the cancer is colorectal cancer. In some instances,the cancer is ovarian cancer. In certain embodiments, the cancer issquamous cell cancer, small-cell lung cancer, non-small cell lungcancer, gastrointestinal cancer, Hodgkin's or non-Hodgkin's lymphoma,pancreatic cancer, glioblastoma, glioma, cervical cancer, ovariancancer, liver cancer, bladder cancer, breast cancer, colon cancer,colorectal cancer, endometrial carcinoma, myeloma, salivary glandcarcinoma, kidney cancer, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, or a type of head or neck cancer. In certain embodiments, thecancer treated is desmoplastic melanoma, inflammatory breast cancer,thymoma, rectal cancer, anal cancer, or surgically treatable ornon-surgically treatable brain stem glioma. In some embodiments, thesubject is a human.

FFPE samples generally are heavily cross-linked and fragmented, andtherefore this type of sample allows for limited RNA recovery usingconventional detection techniques. In certain embodiments, methods oftargeted RNA capture provided herein are less affected by RNAdegradation associated with FFPE fixation than other methods (e.g.,methods that take advantage of oligo-dT capture and reversetranscription of mRNA). In certain embodiments, methods provided hereinenable sensitive measurement of specific genes of interest thatotherwise might be missed with a whole transcriptomic approach.

In some instances, FFPE samples are stained (e.g., using H&E). Themethods disclosed herein are compatible with H&E will allow formorphological context overlaid with transcriptomic analysis. However,depending on the need some samples may be stained with only a nuclearstain, such as staining a sample with only hematoxylin and not eosin,when location of a cell nucleus is needed.

In some embodiments, a biological sample (e.g., tissue section) can befixed with methanol, stained with hematoxylin and eosin, and imaged. Insome embodiments, fixing, staining, and imaging occurs before one ormore probes are hybridized to the sample. Some embodiments of any of theworkflows described herein can further include a destaining step (e.g.,a hematoxylin and eosin destaining step), after imaging of the sampleand prior to permeabilizing the sample. For example, destaining can beperformed by performing one or more (e.g., one, two, three, four, orfive) washing steps (e.g., one or more (e.g., one, two, three, four, orfive) washing steps performed using a buffer including HCl). The imagescan be used to map spatial gene expression patterns back to thebiological sample. A permeabilization enzyme can be used to permeabilizethe biological sample directly on the slide.

In some embodiments, the FFPE sample is deparaffinized, permeabilized,equilibrated, and blocked before target probe oligonucleotides areadded. In some embodiments, deparaffinization using xylenes. In someembodiments, deparaffinization includes multiple washes with xylenes. Insome embodiments, deparaffinization includes multiple washes withxylenes followed by removal of xylenes using multiple rounds of gradedalcohol followed by washing the sample with water. In some aspects, thewater is deionized water. In some embodiments, equilibrating andblocking includes incubating the sample in a pre-Hyb buffer. In someembodiments, the pre-Hyb buffer includes yeast tRNA. In someembodiments, permeabilizing a sample includes washing the sample with aphosphate buffer. In some embodiments, the buffer is PBS. In someembodiments, the buffer is PBST.

(n) Determining the Sequence of the Ligation Product

After an analyte (e.g., mRNA molecule) or a ligation product from thesample has hybridized or otherwise been associated with a capture probeaccording to any of the methods described above in connection with thegeneral spatial cell-based analytical methodology, the barcodedconstructs that result from hybridization/association are analyzed.

In some embodiments, after contacting a biological sample with asubstrate that includes capture probes, a removal step can optionally beperformed to remove all or a portion of the biological sample from thesubstrate. In some embodiments, the removal step includes enzymaticand/or chemical degradation of cells of the biological sample. Forexample, the removal step can include treating the biological samplewith an enzyme (e.g., a proteinase, e.g., proteinase K) to remove atleast a portion of the biological sample from the substrate. In someembodiments, the removal step can include ablation of the tissue (e.g.,laser ablation).

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample), the method comprising: (a) optionally stainingand/or imaging a biological sample on a substrate; (b) permeabilizing(e.g., providing a solution comprising a permeabilization reagent to)the biological sample on the substrate; (c) contacting the biologicalsample with an array comprising a plurality of capture probes, wherein acapture probe of the plurality captures the biological analyte; (d)hybridizing an undesirable RNA depletion probe to an undesirable RNA;(e) removing the plurality of undesirable RNA depletionprobe-undesirable RNA complexes; (f) hybridizing the analyte to acapture domain of a capture probe that is affixed to the substrate; and(g) analyzing the captured biological analyte, thereby spatiallydetecting the biological analyte; wherein the biological sample is fullyor partially removed from the substrate.

In some embodiments, a biological sample is not removed from thesubstrate. For example, the biological sample is not removed from thesubstrate prior to releasing a capture probe (e.g., a capture probebound to an analyte) from the substrate. In some embodiments, suchreleasing comprises cleavage of the capture probe from the substrate(e.g., via a cleavage domain). In some embodiments, such releasing doesnot comprise releasing the capture probe from the substrate (e.g., acopy of the capture probe bound to an analyte can be made and the copycan be released from the substrate, e.g., via denaturation). In someembodiments, the biological sample is not removed from the substrateprior to analysis of an analyte bound to a capture probe after it isreleased from the substrate. In some embodiments, the biological sampleremains on the substrate during removal of a capture probe from thesubstrate and/or analysis of an analyte bound to the capture probe afterit is released from the substrate. In some embodiments, the biologicalsample remains on the substrate during removal (e.g., via denaturation)of a copy of the capture probe (e.g., complement). In some embodiments,analysis of an analyte bound to capture probe from the substrate can beperformed without subjecting the biological sample to enzymatic and/orchemical degradation of the cells (e.g., permeabilized cells) orablation of the tissue (e.g., laser ablation).

In some embodiments, at least a portion of the biological sample is notremoved from the substrate. For example, a portion of the biologicalsample can remain on the substrate prior to releasing a capture probe(e.g., a capture prove bound to an analyte) from the substrate and/oranalyzing an analyte bound to a capture probe released from thesubstrate. In some embodiments, at least a portion of the biologicalsample is not subjected to enzymatic and/or chemical degradation of thecells (e.g., permeabilized cells) or ablation of the tissue (e.g., laserablation) prior to analysis of an analyte bound to a capture probe fromthe substrate.

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample) that include: (a) optionally staining and/or imaginga biological sample on a substrate; (b) permeabilizing (e.g., providinga solution comprising a permeabilization reagent to) the biologicalsample on the substrate; (c) contacting the biological sample with anarray comprising a plurality of capture probes, wherein a capture probeof the plurality captures the biological analyte; (d) hybridizing anundesirable RNA depletion probe to an undesirable RNA; (e) removing theplurality of undesirable RNA depletion probe-undesirable RNA complexes;(f) hybridizing the analyte to a capture domain of a capture probe thatis affixed to the substrate; and (g) analyzing the captured biologicalanalyte, thereby spatially detecting the biological analyte; where thebiological sample is not removed from the substrate.

In some embodiments, provided herein are methods for spatially detectinga biological analyte of interest from a biological sample that include:(a) staining and imaging a biological sample on a substrate; (b)providing a solution comprising a permeabilization reagent to thebiological sample on the substrate; (c) contacting the biological samplewith an array on a substrate, wherein the array comprises one or morecapture probe pluralities thereby allowing the one or more pluralitiesof capture probes to capture the biological analyte of interest; (d)hybridizing an undesirable RNA depletion probe to an undesirable RNA;(e) removing the plurality of undesirable RNA depletionprobe-undesirable RNA complexes; (f) hybridizing the analyte to acapture domain of a capture probe that is affixed to the substrate; and(g) analyzing the captured biological analyte, thereby spatiallydetecting the biological analyte of interest; where the biologicalsample is not removed from the substrate.

In some embodiments, the method further includes subjecting a region ofinterest in the biological sample to spatial transcriptomic analysis. Insome embodiments, one or more of the capture probes includes a capturedomain. In some embodiments, one or more of the capture probes comprisesa unique molecular identifier (UMI). In some embodiments, one or more ofthe capture probes comprises a cleavage domain. In some embodiments, thecleavage domain comprises a sequence recognized and cleaved byuracil-DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease (APE1),U uracil-specific excision reagent (USER), and/or an endonuclease VIII.In some embodiments, one or more capture probes do not comprise acleavage domain and is not cleaved from the array.

In some embodiments, a capture probe can be extended (an “extendedcapture probe,” e.g., as described herein). For example, extending acapture probe can include generating cDNA from a captured (hybridized)RNA. This process involves synthesis of a complementary strand of thehybridized nucleic acid, e.g., generating cDNA based on the captured RNAtemplate (the RNA hybridized to the capture domain of the captureprobe). Thus, in an initial step of extending a capture probe, e.g., thecDNA generation, the captured (hybridized) nucleic acid, e.g., RNA, actsas a template for the extension, e.g., reverse transcription, step. Insome embodiments, the capture probe is extended after hybridizing anundesirable RNA depletion probe to an undesirable RNA and removing theplurality of undesirable RNA depletion probe-undesirable RNA complexes.

In some embodiments, the capture probe is extended using reversetranscription. For example, reverse transcription includes synthesizingcDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), usinga reverse transcriptase. In some embodiments, reverse transcription isperformed while the tissue is still in place, generating an analytelibrary, where the analyte library includes the spatial barcodes fromthe adjacent capture probes. In some embodiments, the capture probe isextended using one or more DNA polymerases.

In some embodiments, a capture domain of a capture probe includes aprimer for producing the complementary strand of a nucleic acidhybridized to the capture probe, e.g., a primer for DNA polymeraseand/or reverse transcription. The nucleic acid, e.g., DNA and/or cDNA,molecules generated by the extension reaction incorporate the sequenceof the capture probe. The extension of the capture probe, e.g., a DNApolymerase and/or reverse transcription reaction, can be performed usinga variety of suitable enzymes and protocols.

In some embodiments, a full-length DNA (e.g., cDNA) molecule isgenerated. In some embodiments, a “full-length” DNA molecule refers tothe whole of the captured nucleic acid molecule. However, if a nucleicacid (e.g., RNA) was partially degraded in the tissue sample, then thecaptured nucleic acid molecules will not be the same length as theinitial RNA in the tissue sample. In some embodiments, the 3′ end of theextended probes, e.g., first strand cDNA molecules, is modified. Forexample, a linker or adaptor can be ligated to the 3′ end of theextended probes. This can be achieved using single stranded ligationenzymes such as T4 RNA ligase or Circligase™ (available from Lucigen,Middleton, WI). In some embodiments, template switching oligonucleotidesare used to extend cDNA in order to generate a full-length cDNA (or asclose to a full-length cDNA as possible). In some embodiments, a secondstrand synthesis helper probe (a partially double stranded DNA moleculecapable of hybridizing to the 3′ end of the extended capture probe), canbe ligated to the 3′ end of the extended probe, e.g., first strand cDNA,molecule using a double stranded ligation enzyme such as T4 DNA ligase.Other enzymes appropriate for the ligation step are known in the art andinclude, e.g., Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain9° N) DNA ligase (9°N™ DNA ligase, New England Biolabs), Ampligase™(available from Lucigen, Middleton, WI), and SplintR (available from NewEngland Biolabs, Ipswich, MA). In some embodiments, a polynucleotidetail, e.g., a poly(A) tail, is incorporated at the 3′ end of theextended probe molecules. In some embodiments, the polynucleotide tailis incorporated using a terminal transferase active enzyme.

In some embodiments, double-stranded extended capture probes are treatedto remove any unextended capture probes prior to amplification and/oranalysis, e.g., sequence analysis. This can be achieved by a variety ofmethods, e.g., using an enzyme to degrade the unextended probes, such asan exonuclease enzyme, or purification columns.

In some embodiments, extended capture probes are amplified to yieldquantities that are sufficient for analysis, e.g., via DNA sequencing.In some embodiments, the first strand of the extended capture probes(e.g., DNA and/or cDNA molecules) acts as a template for theamplification reaction (e.g., a polymerase chain reaction).

In some embodiments, the amplification reaction incorporates an affinitygroup onto the extended capture probe (e.g., RNA-cDNA hybrid) using aprimer including the affinity group. In some embodiments, the primerincludes an affinity group and the extended capture probes includes theaffinity group. The affinity group can correspond to any of the affinitygroups described previously.

In some embodiments, the extended capture probes including the affinitygroup can be coupled to a substrate specific for the affinity group. Insome embodiments, the substrate can include an antibody or antibodyfragment. In some embodiments, the substrate includes avidin orstreptavidin and the affinity group includes biotin. In someembodiments, the substrate includes maltose and the affinity groupincludes maltose-binding protein. In some embodiments, the substrateincludes maltose-binding protein and the affinity group includesmaltose. In some embodiments, amplifying the extended capture probes canfunction to release the extended probes from the surface of thesubstrate, insofar as copies of the extended probes are not immobilizedon the substrate.

In some embodiments, the extended capture probe or complement oramplicon thereof is released. The step of releasing the extended captureprobe or complement or amplicon thereof from the surface of thesubstrate can be achieved in a number of ways. In some embodiments, anextended capture probe or a complement thereof is released from thearray by nucleic acid cleavage and/or by denaturation (e.g., by heatingto denature a double-stranded molecule).

In some embodiments, the extended capture probe or complement oramplicon thereof is released from the surface of the substrate (e.g.,array) by physical means. For example, where the extended capture probeis indirectly immobilized on the array substrate, e.g., viahybridization to a surface probe, it can be sufficient to disrupt theinteraction between the extended capture probe and the surface probe.Methods for disrupting the interaction between nucleic acid moleculesinclude denaturing double stranded nucleic acid molecules are known inthe art. A straightforward method for releasing the DNA molecules (i.e.,of stripping the array of extended probes) is to use a solution thatinterferes with the hydrogen bonds of the double stranded molecules. Insome embodiments, the extended capture probe is released by an applyingheated solution, such as water or buffer, of at least 85° C., e.g., atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99° C. In some embodiments,a solution including salts, surfactants, etc. that can furtherdestabilize the interaction between the nucleic acid molecules is addedto release the extended capture probe from the substrate.

In some embodiments, where the extended capture probe includes acleavage domain, the extended capture probe is released from the surfaceof the substrate by cleavage. For example, the cleavage domain of theextended capture probe can be cleaved by any of the methods describedherein. In some embodiments, the extended capture probe is released fromthe surface of the substrate, e.g., via cleavage of a cleavage domain inthe extended capture probe, prior to the step of amplifying the extendedcapture probe.

In some embodiments, probes complementary to the extended capture probecan be contacted with the substrate. In some embodiments, the biologicalsample can be in contact with the substrate when the probes arecontacted with the substrate. In some embodiments, the biological samplecan be removed from the substrate prior to contacting the substrate withprobes. In some embodiments, the probes can be labeled with a detectablelabel (e.g., any of the detectable labels described herein). In someembodiments, probes that do not specially bind (e.g., hybridize) to anextended capture probe can be washed away. In some embodiments, probescomplementary to the extended capture probe can be detected on thesubstrate (e.g., imaging, any of the detection methods describedherein).

In some embodiments, probes complementary to an extended capture probecan be about 4 nucleotides to about 100 nucleotides long. In someembodiments, probes (e.g., detectable probes) complementary to anextended capture probe can be about 10 nucleotides to about 90nucleotides long. In some embodiments, probes (e.g., detectable probes)complementary to an extended capture probe can be about 20 nucleotidesto about 80 nucleotides long. In some embodiments, probes (e.g.,detectable probes) complementary to an extended capture probe can beabout 30 nucleotides to about 60 nucleotides long. In some embodiments,probes (e.g., detectable probes) complementary to an extended captureprobe can be about 40 nucleotides to about 50 nucleotides long. In someembodiments, probes (e.g., detectable probes) complementary to anextended capture probe can be about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, about 30,about 31, about 32, about 33, about 34, about 35, about 36, about 37,about 38, about 39, about 40, about 41, about 42, about 43, about 44,about 45, about 46, about 47, about 48, about 49, about 50, about 51,about 52, about 53, about 54, about 55, about 56, about 57, about 58,about 59, about 60, about 61, about 62, about 63, about 64, about 65,about 66, about 67, about 68, about 69, about 70, about 71, about 72,about 73, about 74, about 75, about 76, about 77, about 78, about 79,about 80, about 81, about 82, about 83, about 84, about 85, about 86,about 87, about 88, about 89, about 90, about 91, about 92, about 93,about 94, about 95, about 96, about 97, about 98, and about 99nucleotides long.

In some embodiments, about 1 to about 100 probes can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe. In some embodiments, about 1 to about 10 probes can be contactedto the substrate and specifically bind (e.g., hybridize) to an extendedcapture probe. In some embodiments, about 10 to about 100 probes can becontacted to the substrate and specifically bind (e.g., hybridize) to anextended capture probe. In some embodiments, about 20 to about 90 probescan be contacted to the substrate and specifically bind (e.g.,hybridize) to an extended capture probe. In some embodiments, about 30to about 80 probes (e.g., detectable probes) can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe. In some embodiments, about 40 to about 70 probes can be contactedto the substrate and specifically bind (e.g., hybridize) to an extendedcapture probe. In some embodiments, about 50 to about 60 probes can becontacted to the substrate and specifically bind (e.g., hybridize) to anextended capture probe. In some embodiments, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, about 30, about 31, about 32, about33, about 34, about 35, about 36, about 37, about 38, about 39, about40, about 41, about 42, about 43, about 44, about 45, about 46, about47, about 48, about 49, about 50, about 51, about 52, about 53, about54, about 55, about 56, about 57, about 58, about 59, about 60, about61, about 62, about 63, about 64, about 65, about 66, about 67, about68, about 69, about 70, about 71, about 72, about 73, about 74, about75, about 76, about 77, about 78, about 79, about 80, about 81, about82, about 83, about 84, about 85, about 86, about 87, about 88, about89, about 90, about 91, about 92, about 93, about 94, about 95, about96, about 97, about 98, and about 99 probes can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe.

In some embodiments, the probes can be complementary to a single analyte(e.g., a single gene). In some embodiments, the probes can becomplementary to one or more analytes (e.g., analytes in a family ofgenes). In some embodiments, the probes (e.g., detectable probes) can befor a panel of genes associated with a disease (e.g., cancer,Alzheimer's disease, Parkinson's disease).

In some instances, the analyte and capture probe can be amplified orcopied, creating a plurality of cDNA molecules. In some instances, theligated probe and capture probe can be amplified or copied, creating aplurality of cDNA molecules. In some embodiments, cDNA can be denaturedfrom the capture probe template and transferred (e.g., to a clean tube)for amplification, and/or library construction. The spatially-barcodedcDNA can be amplified via PCR prior to library construction. The cDNAcan then be enzymatically fragmented and size-selected in order tooptimize for cDNA amplicon size. P5 and P7 sequences directed tocapturing the amplicons on a sequencing flowcell (Illumina sequencinginstruments) can be appended to the amplicons, i7, and i5 can be used assample indexes, and TruSeq Read 2 can be added via End Repair,A-tailing, Adaptor Ligation, and PCR. The cDNA fragments can then besequenced using paired-end sequencing using TruSeq Read 1 and TruSeqRead 2 as sequencing primer sites. The additional sequences are directedtoward Illumina sequencing instruments or sequencing instruments thatutilize those sequences; however a skilled artisan will understand thatadditional or alternative sequences used by other sequencing instrumentsor technologies are also equally applicable for use in theaforementioned methods.

In some embodiments, where a sample is barcoded directly viahybridization with capture probes or analyte capture agents hybridized,bound, or associated with either the cell surface, or introduced intothe cell, as described above, sequencing can be performed on the intactsample.

A wide variety of different sequencing methods can be used to analyzebarcoded analytes (e.g., an analyte and/or the ligation product). Ingeneral, sequenced polynucleotides can be, for example, nucleic acidmolecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),including variants or derivatives thereof (e.g., single stranded DNA orDNA/RNA hybrids, and nucleic acid molecules with a nucleotide analog).

Sequencing of polynucleotides can be performed by various systems. Moregenerally, sequencing can be performed using nucleic acid amplification,polymerase chain reaction (PCR) (e.g., digital PCR and droplet digitalPCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-basedsingle plex methods, emulsion PCR), and/or isothermal amplification.Non-limiting examples of methods for sequencing genetic materialinclude, but are not limited to, DNA hybridization methods (e.g.,Southern blotting), restriction enzyme digestion methods, Sangersequencing methods, next-generation sequencing methods (e.g.,single-molecule real-time sequencing, nanopore sequencing, and Polonysequencing), ligation methods, and microarray methods.

(o) Kits

In some embodiments, also provided herein are kits that include one ormore reagents to detect one or more analytes described herein. In someinstances, the kit includes a substrate comprising a plurality ofcapture probes comprising a spatial barcode and the capture domain. Insome instances, the kit includes a plurality of probes (e.g., a firstprobe, a second probe, one or more spanning probes, and/or a thirdoligonucleotide).

A non-limiting example of a kit used to perform any of the methodsdescribed herein includes: (a) a substrate comprising a plurality ofcapture probes comprising a spatial barcode and a capture domain; (b) asystem comprising: a plurality of undesirable RNA depletion probes,wherein an undesirable RNA depletion probe of the plurality ofundesirable RNA depletion probes is substantially complementary to asequence of an undesirable RNA molecule in the biological sample; and(c) instructions for performing the method of any one of the precedingclaims.

A non-limiting example of a kit used to perform any of the methodsdescribed herein includes: (a) a substrate comprising a plurality ofcapture probes comprising a spatial barcode and a capture domain; (b) asystem comprising: a first probe oligonucleotide, a second probeoligonucleotide, and a plurality of undesirable RNA depletion probes,wherein the first probe oligonucleotide and the second probeoligonucleotide are substantially complementary to adjacent sequences ofthe analyte, wherein the second probe oligonucleotide comprises acapture probe binding domain that is capable of binding to a capturedomain of a capture probe, and wherein an undesirable RNA depletionprobe of the plurality of undesirable RNA depletion probes issubstantially complementary to a sequence of an undesirable RNA moleculein the biological sample; and (c) instructions for performing the methodof any one of the preceding claims.

In some embodiments of any of the kits described herein, the kitincludes a second probe that includes a preadenylated phosphate group atits 5′ end and a first probe comprising at least two ribonucleic acidbases at the 3′ end.

EXAMPLES Example 1. Workflow of In Situ Spatial RTL (RNA-TemplatedLigation) Ribosomal Depletion

Others have reported ribosomal probe designs and their application toribosomal depletion from purified total RNA. See Morlan et al.,“Selective depletion of rRNA enables whole transcriptome profiling ofarchival fixed tissue.” PloS one 7.8 (2012); US patent applicationpublication No. 20110111409 A1; U.S. patent application No. 62/860,993;and Adiconis et al., “Comparative analysis of RNA sequencing methods fordegraded or low-input samples.” Nature methods 10.7 (2013): 623; each ofwhich is incorporated herein in its entirety by reference. Here, the RNAdepletion procedure is incorporated into the workflow of RNA-templatedligation in a biological sample, wherein the ribosomal RNA for depletionis not purified, but located in a sample, such as a tissue.

As a non-limiting example, and as shown in FIG. 7 , the RNA depletionprocedure can be performed using a biological tissue sample comprisingmRNA and ribosomal RNA (rRNA), where the rRNA is to be depleted from thesample. A plurality of ribosomal depletion probes can be addedsimultaneously to specifically hybridize with rRNA, forming RNA:DNAduplex structures. The ribosomal depletion probes can be designed tohybridize to the complete or partial sequence of the rRNA molecule.After hybridization, RNase H can be added to digest the RNA strand ofthe hybridized RNA:DNA duplex, such that the rRNA can be digested. Thebiological sample can then be permeabilized to release the ligated RTLprobes. In this example, the capture of target mRNA can also beperformed concurrently with the rRNA depletion method. To performconcurrent rRNA depletion and target mRNA capture, two RTL probes (i.e.,LHS and RHS probes) can be applied to the sample simultaneously with therRNA depletion probes. The probes are allowed to hybridize to theirtargets during a hybridization reaction and a ligation step ligates theRTL probes together, followed by RNase H digestion of the RNA of theDNA:RNA formed hybrids, thereby digesting the rRNA and depleting thosemolecules while at the same time releasing the RTL ligation product. Thesample can be permeabilized, thereby contacting the RTL ligationproducts with a plurality of capture probes attached to a slide. Theligated RTL probes can diffuse and bind to a capture probe affixed tothe surface of the slide, wherein the capture probe comprises acomplementary sequence to a sequence on the RHS ligation product. Afterhybridization, the 3′ end of the capture probe can be extended using theligated RTL probes as a template. The extended and ligated RTL probescan then be collected for downstream library preparation and subsequentspatial expression analysis.

As another non-limiting example, RNA depletion probes can be added to abiological sample to specifically hybridize with unwanted RNA molecules.RNase H can then be added to digest the RNA strand of the hybridizedRNA:DNA duplex, such that the unwanted RNA molecules can be digested.The RNA depletion probes can also be removed using RecJ exonuclease. Thebiological sample can then be subjected to a spatial analysis workflowas described herein.

Example 2. In Situ Ribosomal Depletion Increases mRNA Capture withSpatial Transcriptomics in Clinical Samples

In general, ribosomal depletion can be performed by adding rRNA specificprobes before permeabilizing tissue samples. As shown in FIG. 8A,ribosomal depletion probes (RD probes) can specifically hybridize to andinhibit rRNA molecules from non-specifically binding to capture probeson a substrate, thereby increasing mRNA capture with spatialtranscriptomics in clinical samples. After rRNA molecules are removed,the tissue sample can be permeabilized by any permeabilization methodsas described herein. Ribosomal depletion probes were designed to blockcytoplasmic 18S, 28S, 5S and 5.8S rRNA, as well as mitochondrial 16S and12S rRNA. For these set of experiments, the ribosomal depletion probesinclude the nucleic acid sequences of SEQ ID NOs: 1-195. The ribosomaldepletion probes (e.g., SEQ ID NOs: 1-195) were combined into a poolincluding a concentration of 2 μM of each probe in IDTE buffer (10 mMTris, 0.1 mM EDTA, pH 7.5-8.0). For spatial transcriptomic analysis, inthe reverse transcription (RT) step, H₂O (166.3 μl) was replaced with anequivalent volume (166.3 μl) of the pooled ribosomal depletion probes inIDTE buffer. The final concentration of each ribosomal depletion probein the RT reaction mixture was about 1 μM.

As shown in FIGS. 8B-8C, ribosomal depletion using the probes describedherein reduced the 18S rRNA level in the tissue sample. The results ofFIGS. 8D-8E also indicated that ribosomal depletion increasedpolyA-specific probe binding to mRNA.

Effects of ribosomal depletion on gene expression were assessed. Thegene expression levels were compared between normal tissue and ribosomaldepleted tissue samples. Mouse olfactory bulb (MOB), childhood braintumor (PNET) and adipose tissues were analyzed and results are shown inFIGS. 9A-9C, respectively. The results show that most genes exhibitedsimilar expression levels upon ribosomal depletion, as indicated by theR² values. MT-RNR1 and MT-RNR2, which encodes mitochondrial 12S and 16SrRNA, respectively, exhibited reduced expression levels in ribosomaldepleted tissue samples.

Tissue plots indicating the gene expression levels of mitochondrial 12Sand 16S rRNA are shown in FIG. 10 . Both rRNA molecules presented areduced expression level upon ribosomal depletion in a tissue. As shownin FIGS. 11-12 , more UMIs per gene, as well as an increased detectionrate, were observed with in tissue ribosomal depletion in adipose (fat),mouse olfactory bulb (MOB), MOB-181218, and childhood brain cancer(PNET) tissues.

Spatial expression patterns of different Seurat clusters were comparedbetween a normal tissue and a ribosomal depleted tissue in FIG. 13A andFIG. 13B, respectively. The results show that the ribosomal depletedtissue samples exhibited more clear patterns than the normal tissuesamples (see, e.g., FIGS. 13A-B). Analysis of additional normal tissueand ribosomal depleted tissue in FIGS. 14A-14B show that in a tSNE ploteach Seurat cluster from the ribosomal depleted tissue presented clearerboundaries between Seurat clusters as compared to normal tissue samples,indicating an improved dataset quality. The spatial expression patternsfor each of the Seurat clusters from FIG. 14A (normal tissue) and FIG.14B (ribosomal depleted tissue) are shown in FIG. 15 and FIG. 16 ,respectively. These results indicate that ribosomal depletion improvedthe overall analyzing capability and accuracy of the spatial geneexpression analysis methods as described herein.

Another example is shown in FIGS. 17A-17B and FIGS. 18A-18B, whichfurther supported the conclusions above. In the normal tissue sample(see FIGS. 17A-17B), clusters 1 and 5 (indicated by arrows) had highexpression levels on substantially separate regions of the tissue sample(FIG. 17A). However, these two clusters present interpenetrated patternsin the tSNE plot (see FIG. 17B, indicated by arrows). In contrast,clusters 3 and 4 (indicated by arrows) in the ribosomal depleted tissuesample (see FIG. 18A) also had separate expression patterns, butpresented a clearer boundary in the tSNE plot (FIG. 18B, indicated byarrows). Thus, ribosomal depletion improved the overall dataset qualityto reflect a more accurate spatial gene expression pattern.

An additional example of using ribosomal depletion probes in a spatialtranscriptomic workflow is shown in FIGS. 19-21 , which provides datafor both global gene expression and an exemplary set of individualgenes, comparing the control samples to ribosomal depleted samples. Asnoted above, each of the 195 ribosomal depletion probes (e.g., theribosomal depletion probes of SEQ ID NOs: 1-195) were included at 1 μMfinal concentration in the spatial transcriptomics RT reaction mix. Asshown in FIGS. 19A-19D, ribosomal depletion improved detection of anexemplary subset of mRNA molecules, including Perk, Doc2g, and Kctd12,FIGS. 19B-19D, respectively. As the pool of ribosomal depletion probesincluded probes targeting MT-RNR1 and MT-RNR2, depletion of undesirableRNAs was confirmed by comparing detection of housekeeping genes (e.g.,Actb and Gapdh) with detection of MT-RNR1 and MT-RNR2. As shown in FIGS.20A-20D, there was no change in detection of the housekeeping genes buta reduction in detection of MT-RNR1 and MT-RNR2 when samples wereexposed to the ribosomal depletion probes. Additionally, global geneexpression was not affected by the inclusion of the ribosomal depletionprobes in the spatial transcriptomics workflow. Comparison of globalgene expression between control samples and ribosomal depleted samplesshowed significant correlation for all comparisons (Pearson's r>0.97;p<2.2e-16)). Thus, as noted above, this data shows that ribosomaldepletion probes included in the spatial transcriptomics workflowincreased resolution of spatial gene expression patterns by improvingcapture of mRNA molecules while not limiting analysis of global geneexpression.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

Sequence Listing SEQ ID NO: Oligo Name Sequence (5′ to 3′) 1 AG9327_18_TAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTA 1 CGACTTTTAC 2 AG9328_18_TTCCTCTAGATAGTCAAGTTCGACCGTCTTCTCAGCGCTC 2 CGCCAGGGCC 3 AG9329_18_GTGGGCCGACCCCGGCGGGGCCGATCCGAGGGCCTCACT 3 AAACCATCCAA 4 AG9330_18_TCGGTAGTAGCGACGGGCGGTGTGTACAAAGGGCAGGG 4 ACTTAATCAACG 5 AG9331_18CAAGCTTATGACCCGCACTTACTCGGGAATTCCCTCGTTC _5 ATGGGGAATA 6 AG9332_18ATTGCAATCCCCGATCCCCATCACGAATGGGGTTCAACG _6 GGTTACCCGCG 7 AG9333_18CCTGCCGGCGTAGGGTAGGCACACGCTGAGCCAGTCAGT _7 GTAGCGCGCGT 8 AG9334_18GCAGCCCCGGACATCTAAGGGCATCACAGACCTGTTATT _8 GCTCAATCTCG 9 AG9335_18GGTGGCTGAACGCCACTTGTCCCTCTAAGAAGTTGGGGG _9 ACGCCGACCGC 10 AG9336_18TCGGGGGTCGCGTAACTAGTTAGCATGCCAGAGTCTCGT _10 TCGTTATCGGA 11 AG9337_18ATTAACCAGACAAATCGCTCCACCAACTAAGAACGGCCA _11 TGCACCACCAC 12 AG9338_18CCACGGAATCGAGAAAGAGCTATCAATCTGTCAATCCTG _12 TCCGTGTCCGG 13 AG9339_18GCCGGGTGAGGTTTCCCGTGTTGAGTCAAATTAAGCCGC _13 AGGCTCCACTC 14 AG9340_18CTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCTTT _14 GCAACCATA 15 AG9341_18CTCCCCCCGGAACCCAAAGACTTTGGTTTCCCGGAAGCT _15 GCCCGGCGGGT 16 AG9342_18CATGGGAATAACGCCGCCGCATCGCCGGTCGGCATCGTT _16 TATGGTCGGAA 17 AG9343_18CTACGACGGTATCTGATCGTCTTCGAACCTCCGACTTTCG _17 TTCTTGATTA 18 AG9344_18ATGAAAACATTCTTGGCAAATGCTTTCGCTCTGGTCCGTC _18 TTGCGCCGGT 19 AG9345_18CCAAGAATTTCACCTCTAGCGGCGCAATACGAATGCCCC _19 CGGCCGTCCCT 20 AG9346_18CTTAATCATGGCCTCAGTTCCGAAAACCAACAAAATAGA _20 ACCGCGGTCCT 21 AG9347_18ATTCCATTATTCCTAGCTGCGGTATCCAGGCGGCTCGGGC _21 CTGCTTTGAA 22 AG9348_18CACTCTAATTTTTTCAAAGTAAACGCTTCGGGCCCCGCGG _22 GACACTCAGC 23 AG9349_18TAAGAGCATCGAGGGGGCGCCGAGAGGCAAGGGGCGGG _23 GACGGGCGGTGG 24 AG9350_18CTCGCCTCGCGGCGGACCGCCCGCCCGCTCCCAAGATCC _24 AACTACGAGCT 25 AG9351_18TTTTAACTGCAGCAACTTTAATATACGCTATTGGAGCTGG _25 AATTACCGCG 26 AG9352_18GCTGCTGGCACCAGACTTGCCCTCCAATGGATCCTCGTTA _26 AAGGATTTAA 27 AG9353_18AGTGGACTCATTCCAATTACAGGGCCTCGAAAGAGTCCT _27 GTATTGTTATT 28 AG9354_18TTTCGTCACTACCTCCCCGGGTCGGGAGTGGGTAATTTGC _28 GCGCCTGCTG 29 AG9355_18CCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTC _29 CGGAATCGAA 30 AG9356_18CCCTGATTCCCCGTCACCCGTGGTCACCATGGTAGGCACG _30 GCGACTACCA 31 AG9357_18TCGAAAGTTGATAGGGCAGACGTTCGAATGGGTCGTCGC _31 CGCCACGGG 32 AG9358_18GCGTGCGATCGGCCCGAGGTTATCTAGAGTCACCAAAGC _32 CGCCGGCGCCC 33 AG9359_18GCCCCCCGGCCGGGGCCGGAGAGGGGCTGACCGGGTTGG _33 TTTTGATCTGA 34 AG9360_18TAAATGCACGCATCCCCCCCGCGAAGGGGGTCAGCGCCC _34 GTCGGCATGTA 35 AG9361_18TTAGCTCTAGAATTACCACAGTTATCCAAGTAGGAGAGG _35 AGCGAGCGACC 36 AG9362_18AAAGGAACCATAACTGATTTAATGAGCCATTCGCAGTTT _36 CACTGTACCGG 37 AG9363_18CCGTGCGTACTTAGACATGCATGGCTTAATCTTTGAGACA _37 AGCATATGCT 38 AG9364_18TGGCTTAATCTTTGAGACAAGCATATGCTACTGGCAGGA _38 TCAACCAGGTA 39 AG9365_28GACAAACCCTTGTGTCGAGGGCTGACTTTCAATAGATCG _1 CAGCGAGGGAG 40 AG9366_28CTGCTCTGCTACGTACGAAACCCCGACCCAGAAGCAGGT _2 CGTCTACGAAT 41 AG9367_28GGTTTAGCGCCAGGTTCCCCACGAACGTGCGGTGCGTGA _3 CGGGCGAGGG 42 AG9368_28GCGGCCGCCTTTCCGGCCGCGCCCCGTTTCCCAGGACGA _4 AGGGCACTCCG 43 AG9369_28CACCGGACCCCGGTCCCGGCGCGCGGCGGGGCACGCGCC _5 CTCCCGCGGCG 44 AG9370_28GGGCGCGTGGAGGGGIGGGCGGCCCGCCGGCGGGGACAG _6 GCGGGGGACCG 45 AG9371_28GCTATCCGAGGCCAACCGAGGCTCCGCGGCGCTGCCGTA _7 TCGTTCGCCTG 46 AG9372_28GGCGGGATTCTGACTTAGAGGCGTTCAGTCATAATCCCA _8 CAGATGGTAGC 47 AG9373_28TTCGCCCCATTGGCTCCTCAGCCAAGCACATACACCAAAT _9 GTCTGAACCT 48 AG9374_28GCGGTTCCTCTCGTACTGAGCAGGATTACCATGGCAACA _10 ACACATCATCA 49 AG9375_28GTAGGGTAAAACTAACCTGTCTCACGACGGTCTAAACCC _11 AGCTCACGTTC 50 AG9376_28CCTATTAGTGGGTGAACAATCCAACGCTTGGCGAATTCT _12 GCTTCACAATG 51 AG9377_28ATAGGAAGAGCCGACATCGAAGGATCAAAAAGCGACGT _13 CGCTATGAACGC 52 AG9378_28TTGGCCGCCACAAGCCAGTTATCCCTGTGGTAACTTTTCT _14 GACACCTCCT 53 AG9379_28GCTTAAAACCCAAAAGGTCAGAAGGATCGTGAGGCCCCG _15 CTTTCACGGTC 54 AG9380_28TGTATTCGTACTGAAAATCAAGATCAAGCGAGCTTTTGCC _16 CTTCTGCTCC 55 AG9381_28ACGGGAGGTTTCTGTCCTCCCTGAGCTCGCCTTAGGACAC _17 CTGCGTTACC 56 AG9382_28GTTTGACAGGTGTACCGCCCCAGTCAAACTCCCCACCTG _18 GCACTGTCCCC 57 AG9383_28GGAGCGGGTCGCGCCCGGCCGGGCGGGCGCTTGGCGCCA _19 GAAGCGAGAGC 58 AG9384_28CCCTCGGGCTCGCCCCCCCGCCTCACCGGGTCAGTGAAA _20 AAACGATCAGA 59 AG9385_28GTAGTGGTATTTCACCGGCGGCCCGCAGGGCCGCGGACC _21 CCGCCCCGGGC 60 AG9386_28CCCTCGCGGGGACACCGGGIGGGCGCCGGGGGCCTCCCA _22 CTTATTCTACA 61 AG9387_28CCTCTCATGTCTCTTCACCGTGCCAGACTAGAGTCAAGCT _23 CAACAGGGTC 62 AG9388_28TTCTTTCCCCGCTGATTCCGCCAAGCCCGTTCCCTTGGCT _24 GTGGTTTCGC 63 AG9389_28TGGATAGTAGGTAGGGACAGTGGGAATCTCGTTCATCCA _25 TTCATGCGCGT 64 AG9390_28CACTAATTAGATGACGAGGCATTTGGCTACCTTAAGAGA _26 GTCATAGTTAC 65 AG9391_28TCCCGCCGTTTACCCGCGCTTCATTGAATTTCTTCACTTTG _27 ACATTCAGA 66 AG9392_28GCACTGGGCAGAAATCACATCGCGTCAACACCCGCCGCG _28 GGCCTTCGCGA 67 AG9393_28TGCTTTGTTTTAATTAAACAGTCGGATTCCCCTGGTCCGC _29 ACCAGTTCTA 68 AG9394_28AGTCGGCTGCTAGGCGCCGGCCGAGGCGAGGCGCGCGCG _30 GAACCGCGGCC 69 AG9395_28CCGGGGGCGGACCCGGCGGGIGGGACCGGCCCGCGGCCC _31 CTCCGCCGCCT 70 AG9396_28GCCGCCGCCGCCGCCGCGCGCCGAGGAGGAGGGGGGAA _32 CGGGGGGCGGAC 71 AG9397_28GGGCCGGGIGGGTAGGGCGGGGGGACGAACCGCCCCGCC _33 CCGCCGCCCG 72 AG9398_28CCGACCGCCGCCGCCCGACCGCTCCCGCCCCCAGCGGAC _34 GCGCGCGCGAC 73 AG9399_28CGAGACGTGGGGTGGGGGTGGGGGGCGCGCCGCGCCGC _35 CGCCGGGCTCCC 74 AG9400_28CGGGGGCGGCCGCGACGCCCGCCGCAGCTGGGGCGATCC _36 ACGGGAAGGGC 75 AG9401_28CCGGCTCGCGTCCAGAGTCCGCGCCGCCGCCGGCCCCCC _37 GGGTCCCCGGG 76 AG9402_28GCCCCCCTCGCGGGGACCTGCCCCCGCCGGCCGCCCCGG _38 CGGCCGCCGCG 77 AG9403_28CGGCCCCTGCCGCCCCGACCCTTCTCCCCCCGCCGCGCCC _39 CCACGCGGCG 78 AG9404_28CTCCCCCGGGGAGGGGGGAGGACGGGGAGCGGGGGAGA _40 GAGAGAGAGAGA 79 AG9405_28GGGCGCGGGGTGGGGAGGGAGCGAGCGGCGCGCGCGGG _41 TGGGGCGGGGGA 80 AG9406_28GGGCCGCGAGGGGGGTGCCCCGGGCGTGGGGIGGGCGCG _42 CGCCTCGTCCA 81 AG9407_28GCCGCGGCGCGCGCCCAGCCCCGCTTCGCGCCCCAGCCC _43 GACCGACCCAG 82 AG9408_28CCCTTAGAGCCAATCCTTATCCCGAAGTTACGGATCCGGC _44 TTGCCGACTT 83 AG9409_28CCCTTACCTACATTGTTCCAACATGCCAGAGGCTGTTCAC _45 CTTGGAGACC 84 AG9410_28TGCTGCGGATATGGGTACGGCCCGGCGCGAGATTTACAC _46 CCTCTCCCCCG 85 AG9411_28GATTTTCAAGGGCCAGCGAGAGCTCACCGGACGCCGCCG _47 GAACCGCGACG 86 AG9412_28CTTTCCAAGGCACGGGCCCCTCTCTCGGGGCGAACCCATT _48 CCAGGGCGCC 87 AG9413_28CTGCCCTTCACAAAGAAAAGAGAACTCTCCCCGGGGCTC _49 CCGCCGGCTTC 88 AG9414_28TCCGGGATCGGTCGCGTTACCGCACTGGACGCCTCGCGG _50 CGCCCATCTCC 89 AG9415_28GCCACTCCGGATTCGGGGATCTGAACCCGACTCCCTTTCG _51 ATCGGCCGAG 90 AG9416_28GGCAACGGAGGCCATCGCCCGTCCCTTCGGAACGGCGCT _52 CGCCCATCTCT 91 AG9417_28CAGGACCGACTGACCCATGTTCAACTGCTGTTCACATGG _53 AACCCTTCTCC 92 AG9418_28ACTTCGGCCTTCAAAGTTCTCGTTTGAATATTTGCTACTA _54 CCACCAAGAT 93 AG9419_28CTGCACCTGCGGCGGCTCCACCCGGGCCCGCGCCCTAGG _55 CTTCAAGGCTC 94 AG9420_28ACCGCAGCGGCCCTCCTACTCGTCGCGGCGTAGCGTCCG _56 CGGGGCTCCGG 95 AG9421_28GGGCGGGGAGCGGGGCGTGGGCGGGAGGAGGGGAGGAG _57 GCGTGGG 96 AG9422_28GGGCGGGGGAAGGACCCCACACCCCCGCCGCCGCCGCCG _58 CCGCCGCCCTC 97 AG9423_28CGACGCACACCACACGCGCGCGCGCGCGCGCCGCCCCCG _59 CCGCTCCCGTC 98 AG9424_28CACTCTCGACTGCCGGCGACGGCCGGGTATGGGCCCGAC _60 GCTCCAGCGCC 99 AG9425_28ATCCATTTTCAGGGCTAGTTGATTCGGCAGGTGAGTTGTT _61 ACACACTCCT 100 AG9426_28TAGCGGATTCCGACTTCCATGGCCACCGTCCTGCTGTCTA _62 TATCAACCAA 101 AG9427_28CACCTTTTCTGGGGTCTGATGAGCGTCGGCATCGGGCGCC _63 TTAACCCGGC 102 AG9428_28GTTCGGTTCATCCCGCAGCGCCAGTTCTGCTTACCAAAAG _64 TGGCCCACTA 103 AG9429_28GGCACTCGCATTCCACGCCCGGCTCCACGCCAGCGAGCC _65 GGGCTTCTTAC 104 AG9430_28CCATTTAAAGTTTGAGAATAGGTTGAGATCGTTTCGGCCC _66 CAAGACCTCT 105 AG9431_28AATCATTCGCTTTACCGGATAAAACTGCGTGGCGGGGGT _67 GCGTCGGGTCT 106 AG9432_28GCGAGAGCGCCAGCTATCCTGAGGGAAACTTCGGAGGGA _68 ACCAGCTACTA 107 AG9433_28GATGGTTCGATTAGTCTTTCGCCCCTATACCCAGGTCGGA _69 CGACCGATTT 108 AG9434_28GCACGTCAGGACCGCTACGGACCTCCACCAGAGTTTCCT _70 CTGGCTTCGCC 109 AG9435_28CTGCCCAGGCATAGTTCACCATCTTTCGGGTCCTAACACG _71 TGCGCTCGTG 110 AG9436_28CTCCACCTCCCCGGCGCGGCGGGCGAGACGGGCCGGTGG _72 TGCGCCCTCGG 111 AG9437_28CGGACTGGAGAGGCCTCGGGATCCCACCTCGGCCGGCGA _73 GCGCGCCGGCC 112 AG9438_28TTCACCTTCATTGCGCCACGGCGGCTTTCGTGCGAGCCCC _74 CGACTCGCGC 113 AG9439_28ACGTGTTAGACTCCTTGGTCCGTGTTTCAAGACGGGTCGG _75 GTGGGTAGCC 114 AG9440_28GACGTCGCCGCCGACCCCGTGCGCTCGCTCCGCCGTCCCC _76 CTCTTCGGG 115 AG9441_28GACGCGCGCGTGGCCCCGAGAGAACCTCCCCCGGGCCCG _77 ACGGCGCGACC 116 AG9442_28CGCCCGGGGCGCACTGGGGACAGTCCGCCCCGCCCCCCG _78 ACCCGCGCGCG 117 AG9443_28GCACCCCCCCCGTCGCCGGGGCGGGGGCGCGGGGAGGA _79 GGGGTGGGAGAG 118 AG9444_28CGGTCGCGCCGTGGGAGGGGTGGCCCGGCCCCCCCACGA _80 GGAGACGCCGG 119 AG9445_28CGCGCCCCCGCGGGGGAGACCCCCCTCGCGGGGGATTCC _81 CCGCGGGGGTG 120 AG9446_28GGCGCCGGGAGGGGGGAGAGCGCGGCGACGGGTCTCGC _82 TCCCTCGGCCCC 121 AG9447_28GGGATTCGGCGAGTGCTGCTGCCGGGGGGGCTGTAACAC _83 TCGGGGIGGGT 122 AG9448_28TTCGGTCCCGCCGCCCCCGCCGCCGCCGCCACCGCCGCC _84 GCCGCCGCCGC 123 AG9449_28CCCGACCCGCGCGCCCTCCCGAGGGAGGACGCGGGGCCG _85 GGGGGCGGAGA 124 AG9450_28CGGGGGAGGAGGAGGACGGACGGACGGACGGGGCCCCC _86 CGAGCCACCTTC 125 AG9451_28CCCGCCGGGCCTTCCCAGCCGTCCCGGAGCCGGTCGCGG _87 CGCACCGCCGC 126 AG9452_28GGTGGAAATGCGCCCGGCGGCGGCCGGTCGCCGGTCGGG _88 GGACGGTCCCC 127 AG9453_28CGCCGACCCCACCCCCGGCCCCGCCCGCCCACCCCCGCA _89 CCCGCCGGAGC 128 AG9454_28CCGCCCCCTCCGGGGAGGAGGAGGAGGGGCGGCGGGGG _90 AAGGGAGGGCGG 129 AG9455_28GTGGAGGGGTCGGGAGGAACGGGGGGCGGGAAAGATCC _91 GCCGGGCCGCCG 130 AG9456_28ACACGGCCGGACCCGCCGCCGGGTTGAATCCTCCGGGCG _92 GACTGCGCGGA 131 AG9457_28CCCCACCCGTTTACCTCTTAACGGTTTCACGCCCTCTTGA _93 ACTCTCTCTT 132 AG9458_28CAAAGTTCTTTTCAACTTTCCCTTACGGTACTTGTTGACT _94 ATCGGTCTCG 133 AG9459_28TGCCGGTATTTAGCCTTAGATGGAGTTTACCACCCGCTTT _95 GGGCTGCATT 134 AG9460_28CCCAAGCAACCCGACTCCGGGAAGACCCGGGCGCGCGCC _96 GGCCGCTACCG 135 AG9461_28GCCTCACACCGTCCACGGGCTGGGCCTCGATCAGAAGGA _97 CTTGGGCCCCC 136 AG9462_28CACGAGCGGCGCCGGGGAGCGGGTCTTCCGTACGCCACA _98 TGTCCCGCGCC 137 AG9463_28CCGCGGGGCGGGGATTCGGCGCTGGGCTCTTCCCTGTTC _99 ACTCGCCGTTA 138 AG9464_28CTGAGGGAATCCTGGTTAGTTTCTTTTCCTCCGCTGACTA 100 ATATGCTTAA 139 AG9465_28GACTAATATGCTTAAATTCAGCGGGTCGCCACGTCTGATC 101 TGAGGTCGCG 140 AG9466_5.8AAGCGACGCTCAGACAGGCGTAGCCCCGGGAGGAACCC 1 GGGGCCGCAAGT 141 AG9467_5.8GCGTTCGAAGTGTCGATGATCAATGTGTCCTGCAATTCAC _2 ATTAATTCTC 142 AG9468 5.8GCAGCTAGCTGCGTTCTTCATCGACGCACGAGCCGAGTG _3 ATCCACCGCTA 143 AG9469_16AAACCCTGTTCTTGGGTGGGTGTGGGTATAATACTAAGTT _1 GAGATGATAT 144 AG9470_16CATTTACGGGGGAAGGCGCTTTGTGAAGTAGGCCTTATTT _2 CTCTTGTCCT 145 AG9471_16TTCGTACAGGGAGGAATTTGAANGTAGATAGAAACCGAC _3 CTGGATTACTC 146 AG9472_16CGGTCTGAACTCAGATCACGTAGGACTTTAATCGTTGAA _4 CAAACGAACCT 147 AG9473_16TTAATAGCGGCTGCACCATCGGGATGTCCTGATCCAACA _5 TCGAGGTCGTA 148 AG9474_16AACCCTATTGTTGATATGGACTCTAGAATAGGATTGCGCT _6 GTTATCCCTA 149 AG9475_16GGGTAACTTGTTCCGTTGGTCAAGTTATTGGATCAATTGA _7 GTATAGTAGT 150 AG9476_16TCGCTTTGACTGGTGAAGTCTTAGCATGTACTGCTCGGAG _8 GTTGGGTTCT 151 AG9477_16GCTCCGAGGTCGCCCCAACCGAAATTTTTAATGCAGGTTT _9 GGTAGTTTAG 152 AG9478_16GACCTGTGGGTTTGTTAGGTACTGTTTGCATTAATAAATT _10 AAAGCTCCAT 153 AG9479_16AGGGTCTTCTCGTCTTGCTGTGTTATGCCCGCCTCTTCAC _11 GGGCAGGTCA 154 AG9480_16ATTTCACTGGTTAAAAGTAAGAGACAGCTGAACCCTCGT _12 GGAGCCATTCA 155 AG9481_16TACAGGTCCCTATTTAAGGAACAAGTGATTATGCTACCTT _13 TGCACGGTTA 156 AG9482_16GGGTACCGCGGCCGTTAAACATGTGTCACTGGGCAGGCG _14 GTGCCTCTAAT 157 AG9483_16ACTGGTGATGCTAGAGGTGATGTTTTTGGTAAACAGGCG _15 GGGTAAGATTT 158 AG9484_16GCCGAGTTCCTTTTACTTTTTTTAACCTTTCCTTATGAGCA _16 TGCCTGTGT 159 AG9485_16TGGGTTGACAGTGAGGGTAATAATGACTTGTTGGTTGATT _17 GTAGATATTG 160 AG9486_16GGCTGTTAATTGTCAGTTCAGTGTTTTAATCTGACGCAGG _18 CTTATGCGGA 161 AG9487_16GGAGAATGTTTTCATGTTACTTATACTAACATTAGTTCTT _19 CTATAGGGTG 162 AG9488_16ATAGATTGGTCCAATTGGGTGTGAGGAGTTCAGTTATAT _20 GTTTGGGATTT 163 AG9489_16TTTAGGTAGTGGGTGTTGAGCTTGAACGCTTTCTTAATTG _21 GTGGCTGCTT 164 AG9490_16TTAGGCCTACTATGGGTGTTAAATTTTTTACTCTCTCTAC _22 AAGGTTTTTT 165 AG9491_16CCTAGTGTCCAAAGAGCTGTTCCTCTTTGGACTAACAGTT _23 AAATTTACAA 166 AG9492_16GGGATTTAGAGGGTTCTGTGGGCAAATTTAAAGTTGAAC _24 TAAGATTCTA 167 AG9493_16TCTTGGACAACCAGCTATCACCAGGCTCGGTAGGTTTGTC _25 GCCTCTACCT 168 AG9494_16ATAAATCTTCCCACTATTTTGCTACATAGACGGGTGTGCT _26 CTTTTAGCTG 169 AG9495_16TTCTTAGGTAGCTCGTCTGGTTTCGGGGGTCTTAGCTTTG _27 GCTCTCCTTG 170 AG9496_16CAAAGTTATTTCTAGTTAATTCATTATGCAGAAGGTATAG _28 GGGTTAGTCC 171 AG9497_16TTGCTATATTATGCTTGGTTATAATTTTTCATCTTTCCCTT _29 GCGGTACTA 172 AG9498_16TATCTATTGCGCCAGGTTTCAATTTCTATCGCCTATACTTT _30 ATTTGGGTA 173 AG9499_16AATGGTTTGGCTAAGGTTGTCTGGTAGTAAGGTGGAGTG _31 GGTTTGGGGCT 174 AG9500_12GTTCGTCCAAGTGCACTTTCCAGTACACTTACCATGTTAC _1 GACTTGTCTC 175 AG9501_12CTCTATATAAATGCGTAGGGGTTTTAGTTAAATGTCCTTT _2 GAAGTATACT 176 AG9502_12TGAGGAGGGTGACGGGCGGTGTGTACGCGCTTCAGGGCC _3 CTGTTCAACTA 177 AG9503_12AGCACTCTACTCTTAGTTTACTGCTAAATCCACCTTCGAC _4 CCTTAAGTTT 178 AG9504_12CATAAGGGCTATCGTAGTTTTCTGGGGTAGAAAATGTAG _5 CCCATTTCTTG 179 AG9505_12CCACCTCATGGGCTACACCTTGACCTAACGTCTTTACGTG _6 GGTACTTGCG 180 AG9506_12CTTACTTTGTAGCCTTCATCAGGGTTTGCTGAAGATGGCG _7 GTATATAGGC 181 AG9507_12TGAGCAAGAGGTGGTGAGGTTGATCGGGGTTTATCGATT _8 ACAGAACAGGC 182 AG9508_12TCCTCTAGAGGGATATGAAGCACCGCCAGGTCCTTTGAG _9 TTTTAAGCTGT 183 AG9509_12GGCTCGTAGTGTTCTGGCGAGCAGTTTTGTTGATTTAACT _10 GTTGAGGTTT 184 AG9510_12AGGGCTAAGCATAGTGGGGTATCTAATCCCAGTTTGGGT _11 CTTAGCTATTG 185 AG9511_12TGTGTTCAGATATGTTAAAGCCACTTTCGTAGTCTATTTT _12 GTGTCAACTG 186 AG9512_12GAGTTTTTTACAACTCAGGTGAGTTTTAGCTTTATTGGGG _13 AGGGGGTGAT 187 AG9513_12CTAAAACACTCTTTACGCCGGCTTCTATTGACTTGGGTTA _14 ATCGTGTGAC 188 AG9514_12CGCGGTGGCTGGCACGAAATTGACCAACCCTGGGGTTAG _15 TATAGCTTAGT 189 AG9515_12TAAACTTTCGTTTATTGCTAAAGGTTAATCACTGCTGTTT _16 CCCGTGGG 190 AG9516_12TGTGGCTAGGCTAAGCGTTTTGAGCTGCATTGCTGCGTGC _17 TTGATGCTTG 191 AG9517_12TTCCTTTTGATCGTGGTGATTTAGAGGGTGAACTCACTGG _18 AACGGGGATG 192 AG9518_12CTTGCATGTGTAATCTTACTAAGAGCTAATAGAAAGGCT _19 AGGACCAAACC 193 AG9519_5_AAAGCCTACAGCACCCGGTATTCCCAGGCGGTCTCCCAT 1 CCAAGTACTAA 194 AG9520_5_CCAGGCCCGACCCTGCTTAGCTTCCGAGATCAGACGAGA 2 TCGGGCGCGTT 195 AG9521_5_TTCCGAGATCAGACGAGATCGGGCGCGTTCAGGGTGGTA 3 TGGCCGTAGAC

What is claimed is:
 1. A method for depleting an undesirable RNAmolecule in a biological sample, the method comprising: (a) contactingthe biological sample with a first substrate; (b) adding a plurality ofundesirable RNA depletion probes to the biological sample, wherein anundesirable RNA depletion probe of the plurality of undesirable RNAdepletion probes comprises a sequence that is substantiallycomplementary to a sequence of the undesirable RNA molecule; (c)hybridizing the undesirable RNA depletion probe to the undesirable RNAmolecule, thereby generating an undesirable RNA depletionprobe-undesirable RNA molecule complex; (d) removing the undesirable RNAdepletion probe-undesirable RNA molecule complex to deplete theundesirable RNA molecule in the biological sample; (e) aligning thefirst substrate with a second substrate comprising an array, such thatat least a portion of the biological sample is aligned with at least aportion of the array, wherein the array comprises a plurality of captureprobes and wherein a capture probe of the plurality of capture probescomprises a capture domain; and (f) when the portion of the biologicalsample is aligned with at least the portion of the array, hybridizing anucleic acid analyte or an intermediate agent from the biological sampleto the capture domain of the capture probe.
 2. The method of claim 1,wherein the undesirable RNA depletion probe is a DNA probe.
 3. Themethod of claim 1, wherein the removing step comprises contacting theundesirable RNA depletion probe-undesirable RNA molecule complex with aribonuclease.
 4. The method of claim 3, wherein the ribonuclease isRNase H.
 5. The method of claim 4, wherein the RNase H is RNase H1,RNase H2, or a thermostable RNase H.
 6. The method of claim 1, whereinthe undesirable RNA depletion probe is substantially complementary toall or a portion of the sequence of the undesirable RNA molecule.
 7. Themethod of claim 1, wherein multiple undesirable RNA depletion probes ofthe plurality of undesirable RNA depletion probes hybridize to one ormore undesirable RNA molecules in the biological sample.
 8. The methodof claim 1, wherein the undesirable RNA molecule is a transfer RNA(tRNA), a ribosomal RNA (rRNA), a messenger RNA (mRNA), a mitochondrialRNA, a nuclear RNA, a cytoplasmic RNA, or combinations thereof.
 9. Themethod of claim 8, wherein the undesirable RNA molecule is rRNA.
 10. Themethod of claim 1, wherein the intermediate agent is a connected probe,wherein the connected probe is generated by hybridizing a first probeand a second probe to the nucleic acid analyte in the biological sample,and coupling the first probe and the second probe.
 11. The method ofclaim 10, further comprising releasing the connected probe from thenucleic acid analyte prior to hybridizing to capture domain.
 12. Themethod of claim 11, wherein the releasing comprises treating with RNAse.13. The method of claim 10, wherein the coupling comprises ligating thefirst probe and the second probe with a ligase.
 14. The method of claim1, wherein the nucleic acid analyte is mRNA.
 15. The method of claim 1,wherein the capture probe comprises a spatial barcode.
 16. The method ofclaim 15, further comprising determining (i) all or a part of thesequence of the nucleic acid analyte or the intermediate agenthybridized to the capture domain of the capture probe, or a complementthereof, and (ii) the spatial barcode, or a complement thereof, andusing the determined sequence of (i) and (ii) to identify the locationof the nucleic acid analyte in the biological sample.
 17. The method ofclaim 16, further comprising extending a 3′ end of the capture probeusing the nucleic acid analyte or the intermediate agent as a templateto generate an extended capture probe.
 18. The method of claim 17,further comprising generating a nucleic acid molecule that iscomplementary to all or a part of the extended capture probe.
 19. Themethod of claim 16, wherein the determining (i) and (ii) comprisessequencing.
 20. The method of claim 16, wherein the nucleic acid analyteor the intermediate agent is amplified after hybridization to thecapture domain of the capture probe and prior to determining (i) all orpart of the sequence of the nucleic acid analyte or the intermediateagent hybridized to the capture domain, or a complement thereof, and(ii) the spatial barcode, or a complement thereof.
 21. The method ofclaim 1, wherein the capture probe further comprises one or morefunctional domains, a unique molecular identifier, a cleavage domain, orcombinations thereof.
 22. The method of claim 1, wherein the capturedomain comprises a poly-uridine sequence or a poly-thymidine sequence.23. The method of claim 1, wherein the biological sample is a tissuesection.
 24. The method of claim 1, wherein the biological sample is anFFPE tissue sample.
 25. The method of claim 1, wherein the biologicalsample is decrosslinked.
 26. The method of claim 1, wherein thebiological sample is previously stained using hematoxylin and eosin,immunofluorescence, or immunohistochemistry.
 27. The method of claim 1,further comprising imaging the biological sample on the first substrate.28. The method of claim 1, further comprising contacting the biologicalsample with a permeabilization agent comprising proteinase K or pepsinafter aligning the first substrate with the second substrate.
 29. Themethod of claim 1, wherein the undesirable RNA depletion probe furthercomprises a capture moiety comprising streptavidin, avidin, biotin, or afluorophore, wherein the removing step comprises using a capturemoiety-binding agent that specifically binds to the capture moiety. 30.The method of claim 29, wherein the capture moiety is positioned 5′ or3′ to the sequence of the undesirable RNA depletion probe that issubstantially complementary to the sequence of the undesirable RNAmolecule.